Humanized anti-complement factor bb antibodies and uses thereof

ABSTRACT

Provided herein are humanized anti-factor Bb antibodies, methods of producing the antibodies and methods of using the antibodies.

RELATED APPLICATION

This application claims priority to U.S. provisional application Ser.No. 63/012,590, filed Apr. 20, 2020, the entire content of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present application relates to humanized anti-factor Bb antibodiesand the use of the antibodies.

BACKGROUND

The complement system is part of the innate immune system. Its primaryrole is to “complement” the ability of antibodies and phagocytic cellsto clear harmful pathogens from an organism. The complement systemincludes three separate upstream activation pathways (the classicalpathway, the alternative pathway and the lectin pathway), all convergingon a common terminal pathway. Factor B is a component of the alternativepathway of complement and can be cleaved into factor Ba and factor Bb.Factor B also contains a serine protease (SP) domain, and when activatedit provides the catalytic activity of the alternative pathway C3 and C5convertases. Aberrant activation of the complement system can causedamage to host tissue in a wide variety of pathological settings,ranging from autoimmune disease to organ transplantation. There is stilla need for treating diseases or disorders associated with complementsystem. The present invention addresses this need and others.

SUMMARY

The present disclosure provides, in some aspects, humanized anti-factorBb antibodies, compositions comprising the antibodies, methods ofproducing the antibodies, and methods of using the antibodies, forexample, for treating a complement-mediated disease or disorder. Asshown in the data provided herein, the humanized anti-factor Bbantibodies of the present disclosure have a binding affinity withintwo-fold of that of the parent antibody. Unexpectedly, initial attemptsto humanize the parent mouse anti-factor Bb antibody produced a majorityof variants lacking an acceptable binding affinity. Thus, to producehumanized versions having suitable binding affinities (e.g., to treatcomplement-mediated diseases or disorders), multiple rounds ofhumanization were required. Further, only certain V_(H) and V_(L)domains could be combined to produce an antibody that bound to factor Bbwith an acceptable binding affinity see, e.g., antibodies tested inExample 1, Table 9.

Some aspects of the present disclosure provide a humanized antibody thatbinds specifically to human complement factor Bb protein and comprises aheavy chain variable region (V_(H)) comprising the amino acid sequenceof SEQ ID NO: 19 and a light chain variable region (V_(L)) comprisingthe amino acid sequence of SEQ ID NO: 27.

Other aspects of the present disclosure provide a humanized antibodythat binds specifically to human complement factor Bb protein andcomprises a V_(H) comprising the amino acid sequence of SEQ ID NO: 17and a V_(L) comprising the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the humanized antibody binds specifically to thehuman complement factor Bb protein with an affinity of 10⁻⁶ to10⁻⁹ M.

In some embodiments, the humanized antibody inhibits a complementpathway activity. In some embodiments, the complement pathway activityis the alternative pathway (AP) activity.

In some embodiments, the complement AP activity is selected from thegroup consisting of: AP-mediated terminal membrane attack complex (MAC)deposition, AP-mediated hemolysis, C3 fragment deposition on red bloodcells or other cell types, C3b/Bb-mediated cleavage of C3, andC3bBb3b-mediated cleavage of C5.

In some embodiments, the humanized antibody is a bispecific antibody ora multispecific antibody.

In some embodiments, the humanized antibody is selected from the groupconsisting of an Ig monomer, a Fab fragment, a F(ab′)₂ fragment, a scFv,a scAb, and a Fv.

In some embodiments, the humanized antibody comprises a heavy chainconstant region of the isotype IgG1, IgG2, IgG3, or IgG4.

In some embodiments, the humanized antibody comprises an IgG4 constantregion or a variant thereof.

In some embodiments, the heavy chain constant region comprises an aminoacid sequence that is at least 90% identical to any one of SEQ ID NOs:28-30.

In some embodiments, the humanized antibody comprises a heavy chaincomprising the amino acid sequence of any one of SEQ ID NOs: 32-34 and alight chain comprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments, the humanized antibody comprises a heavy chaincomprising the amino acid sequence of any one of SEQ ID NOs: 36-38 and alight chain comprising the amino acid sequence of SEQ ID NO: 39.

Also provided herein are conjugates comprising a humanized antibody ofthe present disclosure.

In some embodiments, a humanized anti-factor Bb antibody of a conjugatecomprises a V_(H) comprising the amino acid sequence of SEQ ID NO: 19and a V_(L) comprising the amino acid sequence of SEQ ID NO: 27. In someembodiments, a humanized anti-factor Bb antibody of a conjugatecomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:34 and a light chain comprising the amino acid sequence of SEQ ID NO:35.

In some embodiments, a humanized anti-factor Bb antibody of a conjugatecomprises a V_(H) comprising the amino acid sequence of SEQ ID NO: 17and a V_(L) comprising the amino acid sequence of SEQ ID NO: 26. In someembodiments, a humanized anti-factor Bb antibody of a conjugatecomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:38 and a light chain comprising the amino acid sequence of SEQ ID NO:39.

Further provided herein are pharmaceutical compositions comprising ahumanized antibody described herein or a conjugate described herein.

In some embodiments, a pharmaceutical composition comprises a humanizedanti-factor Bb antibody that comprises a V_(H) comprising the amino acidsequence of SEQ ID NO: 19 and a V_(L) comprising the amino acid sequenceof SEQ ID NO: 27. In some embodiments, a pharmaceutical compositioncomprises a humanized anti-factor Bb antibody that comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 34 and a lightchain comprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments, a pharmaceutical composition comprises a humanizedanti-factor Bb antibody that comprises a V_(H) comprising the amino acidsequence of SEQ ID NO: 17 and a V_(L) comprising the amino acid sequenceof SEQ ID NO: 26. In some embodiments, a pharmaceutical compositioncomprises a humanized anti-factor Bb antibody that comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 38 and a lightchain comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, a pharmaceutical composition comprises a conjugatethat comprises a humanized anti-factor Bb antibody that comprises aV_(H) comprising the amino acid sequence of SEQ ID NO: 19 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 27. In someembodiments, a pharmaceutical composition comprises a conjugate thatcomprises a humanized anti-factor Bb antibody that comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 34 and a lightchain comprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments, a pharmaceutical composition comprises a conjugatethat comprises a humanized anti-factor Bb antibody that comprises aV_(H) comprising the amino acid sequence of SEQ ID NO: 17 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 26. In someembodiments, a pharmaceutical composition comprises a conjugate thatcomprises a humanized anti-factor Bb antibody that comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 38 and a lightchain comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, a pharmaceutical composition further comprises apharmaceutically acceptable excipient.

Also provided herein are devices comprising a humanized antibodydescribed herein, a conjugate described herein, or a pharmaceuticalcomposition described herein.

In some embodiments, a device comprises a humanized anti-factor Bbantibody that comprises a V_(H) comprising the amino acid sequence ofSEQ ID NO: 19 and a V_(L) comprising the amino acid sequence of SEQ IDNO: 27. In some embodiments, a device comprises a humanized anti-factorBb antibody that comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 34 and a light chain comprising the amino acidsequence of SEQ ID NO: 35.

In some embodiments, a device comprises a humanized anti-factor Bbantibody that comprises a V_(H) comprising the amino acid sequence ofSEQ ID NO: 17 and a V_(L) comprising the amino acid sequence of SEQ IDNO: 26. In some embodiments, a device comprises a humanized anti-factorBb antibody that comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 38 and a light chain comprising the amino acidsequence of SEQ ID NO: 39.

In some embodiments, a device comprises a conjugate that comprises ahumanized anti-factor Bb antibody that comprises a V_(H) comprising theamino acid sequence of SEQ ID NO: 19 and a V_(L) comprising the aminoacid sequence of SEQ ID NO: 27. In some embodiments, a device comprisesa conjugate that comprises a humanized anti-factor Bb antibody thatcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:34 and a light chain comprising the amino acid sequence of SEQ ID NO:35.

In some embodiments, a device comprises a conjugate that comprises ahumanized anti-factor Bb antibody that comprises a V_(H) comprising theamino acid sequence of SEQ ID NO: 17 and a V_(L) comprising the aminoacid sequence of SEQ ID NO: 26. In some embodiments, a device comprisesa conjugate that comprises a humanized anti-factor Bb antibody thatcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:38 and a light chain comprising the amino acid sequence of SEQ ID NO:39.

In some embodiments, a device comprises a pharmaceutical compositionthat comprises a humanized anti-factor Bb antibody that comprises aV_(H) comprising the amino acid sequence of SEQ ID NO: 19 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 27. In someembodiments, a device comprises a pharmaceutical composition thatcomprises a humanized anti-factor Bb antibody that comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 34 and a lightchain comprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments, a device comprises a pharmaceutical compositionthat comprises a humanized anti-factor Bb antibody that comprises aV_(H) comprising the amino acid sequence of

SEQ ID NO: 17 and a V_(L) comprising the amino acid sequence of SEQ IDNO: 26. In some embodiments, a device comprises a pharmaceuticalcomposition that comprises a humanized anti-factor Bb antibody thatcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:38 and a light chain comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the device is an injectable device, forexample, a syringe, a pen, or an electronic injection device (e-Device).

Yet other aspects of the present disclosure provide methods of treatinga subject having a complement-mediated disease or disorder, the methodscomprising administering to the subject an effective amount of ahumanized antibody described herein, a conjugate described herein, or apharmaceutical composition described herein to treat thecomplement-mediated disease or disorder.

In some embodiments, a method of treating a subject having acomplement-mediated disease or disorder comprises administering to thesubject an effective amount of a humanized anti-factor Bb antibody thatcomprises a V_(H) comprising the amino acid sequence of SEQ ID NO: 19and a V_(L) comprising the amino acid sequence of SEQ ID NO: 27, totreat the complement-mediated disease or disorder. In some embodiments,a method of treating a subject having a complement-mediated disease ordisorder comprises administering to the subject an effective amount of ahumanized anti-factor Bb antibody that comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 34 and a light chaincomprising the amino acid sequence of SEQ ID NO: 35, to treat thecomplement-mediated disease or disorder.

In some embodiments, a method of treating a subject having acomplement-mediated disease or disorder comprises administering to thesubject an effective amount of a humanized anti-factor Bb antibody thatcomprises a V_(H) comprising the amino acid sequence of SEQ ID NO: 17and a V_(L) comprising the amino acid sequence of SEQ ID NO: 26, totreat the complement-mediated disease or disorder. In some embodiments,a method of treating a subject having a complement-mediated disease ordisorder comprises administering to the subject an effective amount of ahumanized anti-factor Bb antibody that comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 38 and a light chaincomprising the amino acid sequence of SEQ ID NO: 39, to treat thecomplement-mediated disease or disorder.

In some embodiments, a method of treating a subject having acomplement-mediated disease or disorder comprises administering to thesubject an effective amount of a conjugate that comprises a humanizedanti-factor Bb antibody that comprises a V_(H) comprising the amino acidsequence of SEQ ID NO: 19 and a V_(L) comprising the amino acid sequenceof SEQ ID NO: 27, to treat the complement-mediated disease or disorder.In some embodiments, a method of treating a subject having acomplement-mediated disease or disorder comprises administering to thesubject an effective amount of a conjugate that comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 34 and a light chaincomprising the amino acid sequence of SEQ ID NO: 35, to treat thecomplement-mediated disease or disorder.

In some embodiments, a method of treating a subject having acomplement-mediated disease or disorder comprises administering to thesubject an effective amount of a conjugate that comprises a V_(H)comprising the amino acid sequence of SEQ ID NO: 17 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 26, to treat thecomplement-mediated disease or disorder. In some embodiments, a methodof treating a subject having a complement-mediated disease or disordercomprises administering to the subject an effective amount of aconjugate that comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 38 and a light chain comprising the amino acidsequence of SEQ ID NO: 39, to treat the complement-mediated disease ordisorder. In some embodiments, a method of treating a subject having acomplement-mediated disease or disorder comprises administering to thesubject an effective amount of a pharmaceutical composition thatcomprises a humanized anti-factor Bb antibody that comprises a V_(H)comprising the amino acid sequence of SEQ ID NO: 19 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 27, to treat thecomplement-mediated disease or disorder. In some embodiments, a methodof treating a subject having a complement-mediated disease or disordercomprises administering to the subject an effective amount of apharmaceutical composition that comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 34 and a light chain comprising theamino acid sequence of SEQ ID NO: 35, to treat the complement-mediateddisease or disorder.

In some embodiments, a method of treating a subject having acomplement-mediated disease or disorder comprises administering to thesubject an effective amount of a pharmaceutical composition thatcomprises a V_(H) comprising the amino acid sequence of SEQ ID NO: 17and a V_(L) comprising the amino acid sequence of SEQ ID NO: 26, totreat the complement-mediated disease or disorder. In some embodiments,a method of treating a subject having a complement-mediated disease ordisorder comprises administering to the subject an effective amount of apharmaceutical composition that comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 38 and a light chain comprising theamino acid sequence of SEQ ID NO: 39, to treat the complement-mediateddisease or disorder.

In some embodiments, the complement-mediated disease is selected fromthe group consisting of: IgA nephropathy (Berger's disease), atypicalhemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria(PNH), idiopathic thrombocytopenic purpura (ITP), thromboticthrombocytopenic purpura (TTP), lupus nephritis, ANCA vasculitis,membranous nephropathy, C3 glomerulonephritis (C3GN), focal segmentalglomerulosclerosis (FSGS), multiple sclerosis, macular degeneration,age-related macular degeneration (AMD), rheumatoid arthritis,antiphospholipid antibody syndrome, asthma, ischemia-reperfusion injury,Type II membranoproliferative glomerulonephritis (GN), spontaneous fetalloss, Pauci-immune vasculitis, epidermolysis bullosa, recurrent fetalloss, and traumatic brain injury.

Still other aspects of the present disclosure provide methods ofinhibiting a complement pathway activity in a subject. In someembodiments, the complement pathway activity is an alternative pathway(AP) activity. In some embodiments, a method of inhibiting a complementpathway activity (e.g., AP activity) in a subject comprisesadministering to the subject an effective amount of a humanizedanti-factor Bb antibody, a conjugate comprising a humanized anti-factorBb antibody, or a pharmaceutical composition comprising a humanizedanti-factor Bb antibody, to inhibit the complement pathway activity.

In some embodiments, a method of inhibiting a complement pathwayactivity (e.g., AP activity) in a subject comprises administering to thesubject an effective amount of a humanized anti-factor Bb antibody thatcomprises a V_(H) comprising the amino acid sequence of SEQ ID NO: 19and a V_(L) comprising the amino acid sequence of SEQ ID NO: 27, toinhibit the complement pathway activity. In some embodiments, a methodof inhibiting a complement pathway activity (e.g., AP activity) in asubject comprises administering to the subject an effective amount of ahumanized anti-factor Bb antibody that comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 34 and a light chaincomprising the amino acid sequence of SEQ ID NO: 35, to inhibit thecomplement pathway activity.

In some embodiments, a method of inhibiting a complement pathwayactivity (e.g., AP activity) in a subject comprises administering to thesubject an effective amount of a humanized anti-factor Bb antibody thatcomprises a V_(H) comprising the amino acid sequence of SEQ ID NO: 17and a V_(L) comprising the amino acid sequence of SEQ ID NO: 26, toinhibit the complement pathway activity. In some embodiments, a methodof inhibiting a complement pathway activity (e.g., AP activity) in asubject comprises administering to the subject an effective amount of ahumanized anti-factor Bb antibody that comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 38 and a light chaincomprising the amino acid sequence of SEQ ID NO: 39, to inhibit thecomplement pathway activity.

In some embodiments, a method of inhibiting a complement pathwayactivity (e.g., AP activity) in a subject comprises administering to thesubject an effective amount of a conjugate that comprises a humanizedanti-factor Bb antibody that comprises a V_(H) comprising the amino acidsequence of SEQ ID NO: 19 and a V_(L) comprising the amino acid sequenceof SEQ ID NO: 27, to inhibit the complement pathway activity. In someembodiments, a method of inhibiting a complement pathway activity (e.g.,AP activity) in a subject comprises administering to the subject aneffective amount of a conjugate that comprises a humanized anti-factorBb antibody that comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 34 and a light chain comprising the amino acidsequence of SEQ ID NO: 35, to inhibit the complement pathway activity.

In some embodiments, a method of inhibiting a complement pathwayactivity (e.g., AP activity) in a subject comprises administering to thesubject an effective amount of a conjugate that comprises a humanizedanti-factor Bb antibody that comprises a V_(H) comprising the amino acidsequence of SEQ ID NO: 17 and a V_(L) comprising the amino acid sequenceof SEQ ID NO: 26, to inhibit the complement pathway activity. In someembodiments, a method of inhibiting a complement pathway activity (e.g.,AP activity) in a subject comprises administering to the subject aneffective amount of a conjugate that comprises a humanized anti-factorBb antibody that comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 38 and a light chain comprising the amino acidsequence of SEQ ID NO: 39, to inhibit the complement pathway activity.

In some embodiments, a method of inhibiting a complement pathwayactivity (e.g., AP activity) in a subject comprises administering to thesubject an effective amount of a pharmaceutical composition thatcomprises a humanized anti-factor Bb antibody that comprises a V_(H)comprising the amino acid sequence of SEQ ID NO: 19 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 27, to inhibit thecomplement pathway activity. In some embodiments, a method of inhibitinga complement pathway activity (e.g., AP activity) in a subject comprisesadministering to the subject an effective amount of a pharmaceuticalcomposition that comprises a humanized anti-factor Bb antibody thatcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:34 and a light chain comprising the amino acid sequence of SEQ ID NO:35, to inhibit the complement pathway activity.

In some embodiments, a method of inhibiting a complement pathwayactivity (e.g., AP activity) in a subject comprises administering to thesubject an effective amount of a pharmaceutical composition thatcomprises a humanized anti-factor Bb antibody that comprises a V_(H)comprising the amino acid sequence of SEQ ID NO: 17 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 26, to inhibit thecomplement pathway activity. In some embodiments, a method of inhibitinga complement pathway activity (e.g., AP activity) in a subject comprisesadministering to the subject an effective amount of a pharmaceuticalcomposition that comprises a humanized anti-factor Bb antibody thatcomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:38 and a light chain comprising the amino acid sequence of SEQ ID NO:39, to inhibit the complement pathway activity.

In some embodiments, the complement AP activity is selected from thegroup consisting of: AP-mediated terminal membrane attack complex (MAC)deposition, AP-mediated hemolysis, C3 fragment deposition on red bloodcells or other cell types, C3b/Bb-mediated cleavage of C3, andC3bBb3b-mediated cleavage of C5. In some embodiments, the subject has acomplement-mediated disease or disorder.

In some embodiments, a method further comprises administering to thesubject a therapeutic agent.

In some embodiments, administering is intravenous, subcutaneous, orintramuscular.

Also provided herein are humanized anti-factor Bb antibodies for use ina method for treating a complement-mediated disease or disorder. Furtherprovided herein are conjugates comprising a humanized anti-factor Bbantibody for use in a method for treating a complement-mediated diseaseor disorder. Further still provided herein are pharmaceuticalcompositions comprising a humanized anti-factor Bb antibody for use in amethod for treating a complement-mediated disease or disorder. Stillfurther provided herein are devices comprising a humanized anti-factorBb antibody for use in a method for treating a complement-mediateddisease or disorder.

In some embodiments, a humanized anti-factor Bb antibody for use in amethod for treating a complement-mediated disease or disorder comprisesa V_(H) comprising the amino acid sequence of SEQ ID NO: 19 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 27. In someembodiments, a humanized anti-factor Bb antibody for use in a method fortreating a complement-mediated disease or disorder comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 34 and a lightchain comprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments, a humanized anti-factor Bb antibody for use in amethod for treating a complement-mediated disease or disorder comprisesa V_(H) comprising the amino acid sequence of SEQ ID NO: 17 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 26. In someembodiments, a humanized anti-factor Bb antibody for use in a method fortreating a complement-mediated disease or disorder comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 38 and a lightchain comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, a conjugate for use in a method for treating acomplement-mediated disease or disorder comprises a humanizedanti-factor Bb antibody comprising a V_(H) comprising the amino acidsequence of SEQ ID NO: 19 and a V_(L) comprising the amino acid sequenceof SEQ ID NO: 27. In some embodiments, a conjugate for use in a methodfor treating a complement-mediated disease or disorder comprises ahumanized anti-factor Bb antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 34 and a light chain comprisingthe amino acid sequence of SEQ ID NO: 35.

In some embodiments, a conjugate for use in a method for treating acomplement-mediated disease or disorder comprises a humanizedanti-factor Bb antibody comprising a V_(H) comprising the amino acidsequence of SEQ ID NO: 17 and a V_(L) comprising the amino acid sequenceof SEQ ID NO: 26. In some embodiments, a conjugate for use in a methodfor treating a complement-mediated disease or disorder comprises ahumanized anti-factor Bb antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 38 and a light chain comprisingthe amino acid sequence of SEQ ID NO: 39.

In some embodiments, a pharmaceutical composition for use in a methodfor treating a complement-mediated disease or disorder comprises ahumanized anti-factor Bb antibody comprising a V_(H) comprising theamino acid sequence of SEQ ID NO: 19 and a V_(L) comprising the aminoacid sequence of SEQ ID NO: 27. In some embodiments, a pharmaceuticalcomposition for use in a method for treating a complement-mediateddisease or disorder comprises a humanized anti-factor Bb antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 34 and a light chain comprising the amino acid sequence of SEQ IDNO: 35.

In some embodiments, a pharmaceutical composition for use in a methodfor treating a complement-mediated disease or disorder comprises ahumanized anti-factor Bb antibody comprising a V_(H) comprising theamino acid sequence of SEQ ID NO: 17 and a V_(L) comprising the aminoacid sequence of SEQ ID NO: 26. In some embodiments, a pharmaceuticalcomposition for use in a method for treating a complement-mediateddisease or disorder comprises a humanized anti-factor Bb antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 38 and a light chain comprising the amino acid sequence of SEQ IDNO: 39.

In some embodiments, a device for use in a method for treating acomplement-mediated disease or disorder comprises a humanizedanti-factor Bb antibody comprising a V_(H) comprising the amino acidsequence of SEQ ID NO: 19 and a V_(L) comprising the amino acid sequenceof SEQ ID NO: 27. In some embodiments, a device for use in a method fortreating a complement-mediated disease or disorder comprises a humanizedanti-factor Bb antibody comprising a heavy chain comprising the aminoacid sequence of SEQ ID NO: 34 and a light chain comprising the aminoacid sequence of SEQ ID NO: 35.

In some embodiments, a device for use in a method for treating acomplement-mediated disease or disorder comprises a humanizedanti-factor Bb antibody comprising a V_(H) comprising the amino acidsequence of SEQ ID NO: 17 and a V_(L) comprising the amino acid sequenceof SEQ ID NO: 26 In some embodiments, a device for use in a method fortreating a complement-mediated disease or disorder comprises a humanizedanti-factor Bb antibody comprising a heavy chain comprising the aminoacid sequence of SEQ ID NO: 38 and a light chain comprising the aminoacid sequence of SEQ ID NO: 39.

In some embodiments, the complement-mediated disease is selected fromthe group consisting of: IgA nephropathy (Berger's disease), atypicalhemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria(PNH), idiopathic thrombocytopenic purpura (ITP), thromboticthrombocytopenic purpura (TTP), lupus nephritis, ANCA vasculitis,membranous nephropathy, C3 glomerulonephritis (C3GN), focal segmentalglomerulosclerosis (FSGS), multiple sclerosis, macular degeneration,age-related macular degeneration (AMD), rheumatoid arthritis,antiphospholipid antibody syndrome, asthma, ischemia-reperfusion injury,Type II membranoproliferative GN, spontaneous fetal loss, Pauci-immunevasculitis, epidermolysis bullosa, recurrent fetal loss, and traumaticbrain injury.

Also provided herein are humanized anti-factor Bb antibodies for use ina method for inhibiting a complement pathway activity (e.g., APactivity). Further provided herein are conjugates comprising a humanizedanti-factor Bb antibody for use in a method for inhibiting a complementpathway activity (e.g., AP activity). Further still provided herein arepharmaceutical compositions comprising a humanized anti-factor Bbantibody for use in a method for inhibiting a complement pathwayactivity (e.g., AP activity). Still further provided herein are devicescomprising a humanized anti-factor Bb antibody for use in a method forinhibiting a complement pathway activity (e.g., AP activity).

In some embodiments, a humanized anti-factor Bb antibody for use in amethod for inhibiting a complement pathway activity (e.g., AP activity)comprises a V_(H) comprising the amino acid sequence of SEQ ID NO: 19and a V_(L) comprising the amino acid sequence of SEQ ID NO: 27. In someembodiments, a humanized anti-factor Bb antibody for use in a method forinhibiting a complement pathway activity (e.g., AP activity) comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 34 and alight chain comprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments, a humanized anti-factor Bb antibody for use in amethod for inhibiting a complement pathway activity (e.g., AP activity)comprises a V_(H) comprising the amino acid sequence of SEQ ID NO: 17and a V_(L) comprising the amino acid sequence of SEQ ID NO: 26. In someembodiments, a humanized anti-factor Bb antibody for use in a method forinhibiting a complement pathway activity (e.g., AP activity) comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 38 and alight chain comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, a conjugate for use in a method for inhibiting acomplement pathway activity (e.g., AP activity) comprises a humanizedanti-factor Bb antibody comprising a V_(H) comprising the amino acidsequence of SEQ ID NO: 19 and a V_(L) comprising the amino acid sequenceof SEQ ID NO: 27. In some embodiments, a conjugate for use in a methodfor inhibiting a complement pathway activity (e.g., AP activity)comprises a humanized anti-factor Bb antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO: 34 and a light chaincomprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments, a conjugate for use in a method for inhibiting acomplement pathway activity (e.g., AP activity) comprises a humanizedanti-factor Bb antibody comprising a V_(H) comprising the amino acidsequence of SEQ ID NO: 17 and a V_(L) comprising the amino acid sequenceof SEQ ID NO: 26. In some embodiments, a conjugate for use in a methodfor inhibiting a complement pathway activity (e.g., AP activity)comprises a humanized anti-factor Bb antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO: 38 and a light chaincomprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, a pharmaceutical composition for use in a methodfor inhibiting a complement pathway activity (e.g., AP activity)comprises a humanized anti-factor Bb antibody comprising a V_(H)comprising the amino acid sequence of SEQ ID NO: 19 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 27. In someembodiments, a pharmaceutical composition for use in a method forinhibiting a complement pathway activity (e.g., AP activity) comprises ahumanized anti-factor Bb antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 34 and a light chain comprisingthe amino acid sequence of SEQ ID NO: 35.

In some embodiments, a pharmaceutical composition for use in a methodfor inhibiting a complement pathway activity (e.g., AP activity)comprises a humanized anti-factor Bb antibody comprising a V_(H)comprising the amino acid sequence of SEQ ID NO: 17 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 26. In someembodiments, a pharmaceutical composition for use in a method forinhibiting a complement pathway activity (e.g., AP activity) comprises ahumanized anti-factor Bb antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 38 and a light chain comprisingthe amino acid sequence of SEQ ID NO: 39.

In some embodiments, a pharmaceutical composition for use in a methodfor inhibiting a complement pathway activity (e.g., AP activity)comprises a humanized anti-factor Bb antibody comprising a V_(H)comprising the amino acid sequence of SEQ ID NO: 19 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 27. In someembodiments, a pharmaceutical composition for use in a method forinhibiting a complement pathway activity (e.g., AP activity) comprises ahumanized anti-factor Bb antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 34 and a light chain comprisingthe amino acid sequence of SEQ ID NO: 35.

In some embodiments, a pharmaceutical composition for use in a methodfor inhibiting a complement pathway activity (e.g., AP activity)comprises a humanized anti-factor Bb antibody comprising a V_(H)comprising the amino acid sequence of SEQ ID NO: 17 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 26. In someembodiments, a pharmaceutical composition for use in a method forinhibiting a complement pathway activity (e.g., AP activity) comprises ahumanized anti-factor Bb antibody comprising a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 38 and a light chain comprisingthe amino acid sequence of SEQ ID NO: 39.

Also provided herein are nucleic acids or nucleic acid sets encoding orcollectively encoding a humanized antibody described herein; vectors orvector sets comprising a nucleic acid or nucleic acid set describedherein; cells expressing a humanized antibody, a nucleic acid or nucleicacid set, or a vector or vector set as described herein.

In some embodiments, a nucleic acid or nucleic acid set encodes orcollectively encodes a humanized anti-factor Bb antibody that comprisesa V_(H) comprising the amino acid sequence of SEQ ID NO: 19 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 27. In someembodiments, a nucleic acid or nucleic acid set encodes or collectivelyencodes a humanized anti-factor Bb antibody that comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 34 and a light chaincomprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments, a nucleic acid or nucleic acid set encodes orcollectively encodes a humanized anti-factor Bb antibody that comprisesa V_(H) comprising the amino acid sequence of SEQ ID NO: 17 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 26. In someembodiments, a nucleic acid or nucleic acid set encodes or collectivelyencodes a humanized anti-factor Bb antibody that comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 38 and a light chaincomprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, a vector or vector set comprises a nucleic acid ornucleic acid set that encodes or collectively encodes a humanizedanti-factor Bb antibody that comprises a V_(H) comprising the amino acidsequence of SEQ ID NO: 19 and a V_(L) comprising the amino acid sequenceof SEQ ID NO: 27. In some embodiments, a vector or vector set comprisesa nucleic acid or nucleic acid set that encodes or collectively encodesa humanized anti-factor Bb antibody that comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 34 and a light chaincomprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments, a vector or vector set comprises a nucleic acid ornucleic acid set that encodes or collectively encodes a humanizedanti-factor Bb antibody that comprises a V_(H) comprising the amino acidsequence of SEQ ID NO: 17 and a V_(L) comprising the amino acid sequenceof SEQ ID NO: 26. In some embodiments, a vector or vector set comprisesa nucleic acid or nucleic acid set that encodes or collectively encodesa humanized anti-factor Bb antibody that comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 38 and a light chaincomprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, a cell comprises a humanized anti-factor Bbantibody that comprises a V_(H) comprising the amino acid sequence ofSEQ ID NO: 19 and a V_(L) comprising the amino acid sequence of SEQ IDNO: 27. In some embodiments, a cell comprises a humanized anti-factor Bbantibody that comprises a heavy chain comprising the amino acid sequenceof SEQ ID NO: 34 and a light chain comprising the amino acid sequence ofSEQ ID NO: 35.

In some embodiments, a cell comprises a humanized anti-factor Bbantibody that comprises a V_(H) comprising the amino acid sequence ofSEQ ID NO: 17 and a V_(L) comprising the amino acid sequence of SEQ IDNO: 26. In some embodiments, a cell comprises a humanized anti-factor Bbantibody that comprises a heavy chain comprising the amino acid sequenceof SEQ ID NO: 38 and a light chain comprising the amino acid sequence ofSEQ ID NO: 39.

In some embodiments, a cell comprises a nucleic acid or nucleic acid setthat encodes or collectively encodes a humanized anti-factor Bb antibodythat comprises a V_(H) comprising the amino acid sequence of SEQ ID NO:19 and a V_(L) comprising the amino acid sequence of SEQ ID NO: 27. Insome embodiments, a cell comprises a nucleic acid or nucleic acid setthat encodes or collectively encodes a humanized anti-factor Bb antibodythat comprises a heavy chain comprising the amino acid sequence of SEQID NO: 34 and a light chain comprising the amino acid sequence of SEQ IDNO: 35.

In some embodiments, a cell comprises a nucleic acid or nucleic acid setthat encodes or collectively encodes a humanized anti-factor Bb antibodythat comprises a V_(H) comprising the amino acid sequence of SEQ ID NO:17 and a V_(L) comprising the amino acid sequence of SEQ ID NO: 26. Insome embodiments, a cell comprises a nucleic acid or nucleic acid setthat encodes or collectively encodes a humanized anti-factor Bb antibodythat comprises a heavy chain comprising the amino acid sequence of SEQID NO: 38 and a light chain comprising the amino acid sequence of SEQ IDNO: 39.

In some embodiments, a cell comprises a vector or vector set thatcomprises a nucleic acid or nucleic acid set that encodes orcollectively encodes a humanized anti-factor Bb antibody that comprisesa V_(H) comprising the amino acid sequence of SEQ ID NO: 19 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 27. In someembodiments, a cell comprises a vector or vector set that comprises anucleic acid or nucleic acid set that encodes or collectively encodes ahumanized anti-factor Bb antibody that comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 34 and a light chaincomprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments, a cell comprises a vector or vector set thatcomprises a nucleic acid or nucleic acid set that encodes orcollectively encodes a humanized anti-factor Bb antibody that comprisesa V_(H) comprising the amino acid sequence of SEQ ID NO: 17 and a V_(L)comprising the amino acid sequence of SEQ ID NO: 26. In someembodiments, a cell comprises a vector or vector set that comprises anucleic acid or nucleic acid set that encodes or collectively encodes ahumanized anti-factor Bb antibody that comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 38 and a light chaincomprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, a cell is a mammalian cell, for example, selectedfrom the group consisting of human embryonic kidney (HEK) cells, Chinesehamster ovary (CHO) cells, NSO myeloma cells, SP2 cells, COS cells, andmammary epithelial cells.

Other aspects of the present disclosure provide methods of producing ahumanized antibody described herein, the methods comprising culturing acell described herein to produce the humanized antibody. In someembodiments, a method further comprises isolating the humanizedantibody.

In some embodiments, a method of producing a humanized anti-factor Bbantibody comprises culturing a cell that comprises a nucleic acid thatencodes or nucleic acid set that collectively encodes a humanizedanti-factor Bb antibody that comprises a V_(H) comprising the amino acidsequence of SEQ ID NO: 19 and a V_(L) comprising the amino acid sequenceof SEQ ID NO: 27 to produce the humanized anti-factor Bb antibody. Insome embodiments, a method of producing a humanized anti-factor Bbantibody comprises culturing a cell that comprises a nucleic acid thatencodes or nucleic acid set that collectively encodes humanizedanti-factor Bb antibody that comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 34 and a light chain comprising theamino acid sequence of SEQ ID NO: 35.

In some embodiments, a method of producing a humanized anti-factor Bbantibody comprises culturing a cell that comprises a nucleic acid thatencodes or nucleic acid set that collectively encodes a humanizedanti-factor Bb antibody that comprises a V_(H) comprising the amino acidsequence of SEQ ID NO: 17 and a V_(L) comprising the amino acid sequenceof SEQ ID NO: 16 to produce the humanized anti-factor Bb antibody. Insome embodiments, a method of producing a humanized anti-factor Bbantibody comprises culturing a cell that comprises a nucleic acid thatencodes or nucleic acid set that collectively encodes humanizedanti-factor Bb antibody that comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 28 and a light chain comprising theamino acid sequence of SEQ ID NO: 39.

The summary above is meant to illustrate, in a non-limiting manner, someof the embodiments, advantages, features, and uses of the technologydisclosed herein. Other embodiments, advantages, features, and uses ofthe technology disclosed herein will be apparent from the DetailedDescription, the Drawings, the Examples, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings: FIGS. 1A-1D show binding of humanized antibodyvariants (Group 1) to complement factor Bb (factor Bb) using singlecycle kinetics. Raw sensorgrams and fitted curves (1:1 binding model)are shown for humanized variants and control antibodies binding tofactor Bb. Kinetic analysis was carried out on a Biacore T200. Eachantibody was captured on a Protein A CM5 chip before increasingconcentrations of factor Bb were injected and a single off-rate wasdetermined. *Scales are identical for each section, and x is from −200to 1400 and y from −5 to 40.

FIGS. 2A-2C show binding of humanized variants (Group 2) to factor Bbusing single cycle kinetics. Raw sensorgrams and fitted curves (1:1binding model) for redesigned variants and control antibodies binding tofactor Bb are shown. Kinetic analysis was carried out on a Biacore T200.Each antibody was captured on a Protein A CM5 chip before increasingconcentrations of factor Bb were injected and a single off-rate wasdetermined.

FIG. 3 shows SDS-PAGE gels of Protein A/Post-SEC-purified antibodies. 1μg of each reduced antibody sample was loaded on a NuPage 4-12% Bis-Trisgel (ThermoFisher, Loughborough, UK) and run at 200 V for 35 minutes.Gels were stained with InstantBlue (Expedeon, Swavesey, UK). Mk:PAGERuler™ Plus pre-stained protein ladder (ThermoFisher, Loughborough,UK).

FIGS. 4A-4C show binding of lead antibodies (all from Group 2) to factorBb using multi-cycle kinetics. Multiple cycle sensorgrams data andfitted curves (1:1 binding model) are shown for the humanized variantsbinding to factor Bb. (FIG. 4A: Chimeric VH0/Vκ0, VH4/Vκ6; FIG. 4B:VH4/Vκ7, VH6/Vκ6; FIG. 4C: VH6/Vκ7, VH7/Vκ7.

FIG. 5 shows factor Bb competition enzyme-linked immunosorbent assay(ELISA) of humanized variants against parental antibody. A dilutionseries of the anti-factor Bb variants was tested against a fixedconcentration of murine parental antibody for binding to factor-Bb.Bound murine antibody was detected using anti-mouse peroxidase conjugateand tetramethylbenzidine (TMB) substrate.

FIGS. 6A-6C show activity of humanized anti-factor Bb antibodies inWIESLAB® Complement Alternative Pathway (CAP) (FIGS. 6A) and AP-mediatedhemolysis (FIGS. 6B-6C) using human serum.

FIGS. 7A-7D show specificity of lead humanized variants to active form(factor Bb) of human factor B (FIGS. 7A and 7B) or cynomolgus monkeyfactor B (FIGS. 7C and 7D) by surface plasmon resonance.

FIGS. 8A-8B show specificity of binding of chimeric parent antibodyV_(H)0/V_(K)0-IgG4v1 (FIG. 8A) and representative humanized variantantibody V_(H)6/V_(K)7-IgG4v2 (FIG. 8B) to factor Bb only among variouscomplement proteins.

FIGS. 9A-9B show activity of V_(H)6/V_(K)7-IgG4v2 (produced from HEKcells) and V_(H)6/V_(K)7-IgG4v2_CHO (produced from CHO cells) inWIESLAB® Complement Alternative Pathway (CAP) assay using normal human(FIG. 9A) and Cyno (FIG. 9B) serum.

FIGS. 10A-10B show activity of V_(H)6/V_(K)7-IgG4v2 (produced from HEK)and V_(H)6/V_(k)7-IgG4v2_CHO in WIESLAB® Complement Classical Pathway(CCP) assay using normal human (FIG. 10A) and Cyno (FIG. 10B) serum.

FIG. 11 show hemolysis of Rabbit RBC by V_(H)6/V_(K)7-IgG4v2_HEK andV_(H)6/V_(K)7-IgG4v2_CHO in normal human serum.

FIG. 12 show affinity and multiple cycle sensorgrams raw data withfitted curves for V_(H)6/V_(K)7-IgG4v2_CHO(left) andV_(H)6/V_(K)7-IgG4v2_HEK(right) binding to human Bb protein.

DETAILED DESCRIPTION

The present disclosure provides humanized antibodies that bindcomplement factor Bb protein. These antibodies are referred to herein as“humanized anti-factor Bb antibodies.” The present disclosure alsoprovides nucleic acids encoding humanized anti-factor Bb antibodies,compositions comprising the antibodies, methods of producing theantibodies (e.g., recombinant production methods), and methods of usingthe antibodies, such as methods of treating a (at least one)complement-mediated disease or disorder.

“Antibody” encompasses antibodies or immunoglobulins of any isotype,including but not limited to humanized antibodies and chimericantibodies. An antibody may be a single-chain antibody (scAb) or asingle domain antibody (dAb) (e.g., a single domain heavy chain antibodyor a single domain light chain antibody; see Holt et al. (2003) TrendsBiotechnol. 21:484). The term “antibody” also encompasses fragments ofantibodies (antibody fragments) that retain specific binding to anantigen. “Antibody” further includes single-chain variable fragments(scFvs), which are fusion proteins of the variable regions of the heavy(V_(H)) and light chains (V_(L)) of antibodies, connected with a shortlinker peptide, and diabodies, which are noncovalent dimers of scFvfragments that include the V_(H) and V_(L) connected by a small peptidelinker (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)). Otherfusion proteins that comprise an antigen-binding portion of an antibodyand a non-antibody protein are also encompassed by the term “antibody.”

“Antibody fragments” comprise a portion of an intact antibody, forexample, the antigen binding or variable region of the intact antibody.Examples of antibody fragments include an antigen-binding fragment(Fab), Fab′, F(ab′)₂, a variable domain Fv fragment (Fv), an Fdfragment, and an antigen binding fragment of a chimeric antigenreceptor.

Papain digestion of antibodies produces two identical antigen-bindingfragments, referred to as “Fab′” fragments, each with a singleantigen-binding site, and a residual “Fc” fragment, a designationreflecting the ability to crystallize readily. Pepsin treatment yieldsan F(ab′)₂ fragment that has two antigen combining sites and is stillcapable of cross-linking antigen.

“Fv” is the minimum antibody fragment that contains a completeantigen-recognition and -binding site. This region includes a dimer ofone heavy-chain variable domain and one light-chain variable domain intight, non-covalent association. It is in this configuration that thethree CDRs of each variable domain interact to define an antigen-bindingsite on the surface of the V_(H)-V_(L) dimer. Collectively, the six CDRsconfer antigen-binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

“Fab′” fragments contain the constant domain of the light chain and thefirst constant domain (CH₁) of the heavy chain. Fab fragments differfrom Fab′ fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH₁ domain including at least one cysteinefrom the antibody hinge region. Fab′-SH is the designation herein forFab′ in which the cysteine residue(s) of the constant domains bear afree thiol group. F(ab′)₂ antibody fragments originally were produced aspairs of Fab′ fragments which have hinge cysteines between them. Otherchemical couplings of antibody fragments are also known.

“scFv” antibody fragments comprise the V_(H) and V_(L) of an antibody,wherein these regions are present in a single polypeptide chain. In somecases, the Fv polypeptide further comprises a polypeptide linker betweenthe V_(H) and V_(L) regions, which enables the scFv to form the desiredstructure for antigen binding. For a review of scFv, see Pluckthun inThe Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds., Springer-Verlag, New York, pp. 269-315 (1994).

“Diabody” refers to a small antibody fragment with two antigen-bindingsites, which fragments comprise a V_(H) connected to a V_(L) in the samepolypeptide chain (V_(H)-V_(L)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, Hollinger et al. Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993).

An antibody can be monovalent or bivalent. An antibody can be an Igmonomer, which is a “Y-shaped” molecule that consists of fourpolypeptide chains: two heavy chains and two light chains connected bydisulfide bonds.

Antibodies can be detectably labeled, e.g., with a radioisotope, anenzyme that generates a detectable product, and/or a fluorescentprotein. Antibodies can be further conjugated to other moieties, such asmembers of specific binding pairs, e.g., biotin member of biotin-avidinspecific binding pair. Antibodies can also be bound to a solid support,including, but not limited to, polystyrene plates and/or beads.

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment (i.e., isnot naturally occurring). Contaminant components of its naturalenvironment are materials that would interfere with uses (e.g.,diagnostic or therapeutic uses) of the antibody, and can includeenzymes, hormones, and other proteinaceous or nonproteinaceous solutes.In some cases, an antibody is purified (1) to greater than 90%, greaterthan 95%, or greater than 98% by weight of antibody as determined by theLowry method, for example, more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) under reducing or non-reducing conditions using Coomassieblue or silver stain. Isolated antibodies encompass antibodies in situwithin recombinant cells, as at least one component of the antibody'snatural environment will not be present. In some embodiments, anisolated antibody is prepared by at least one purification step.

A “monoclonal antibody” is an antibody produced by a group of identicalcells, all of which were produced from a single cell by repetitivecellular replication. That is, the clone of cells only produces a singleantibody species. While a monoclonal antibody can be produced usinghybridoma production technology, other production methods known to thoseskilled in the art can also be used (e.g., antibodies derived fromantibody phage display libraries).

A “complementarity determining region (CDR)” is the non-contiguousantigen combining sites found within the variable region of both heavyand light chain polypeptides. CDRs have been described by Lefranc et al.(2003) Developmental and Comparative Immunology 27:55; Kabat et al., J.Biol. Chem. 252:6609-6616 (1977); Kabat et al., U. S. Dept. of Healthand Human Services, “Sequences of proteins of immunological interest”(1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987); andMacCallum et al., J. Mol. Biol. 262:732-745 (1996), where thedefinitions include overlapping or subsets of amino acid residues whencompared against each other. Nevertheless, application of eitherdefinition to refer to a CDR of an antibody or grafted antibodies orvariants thereof is intended to be within the scope of the term asdefined and used herein.

As used herein, the terms “CDR-L1,” “CDR-L2,” and “CDR-L3” refer,respectively, to the first, second, and third CDRs in a light chainvariable region. As used herein, the terms “CDR-H1”, “CDR-H2”, and“CDR-H3” refer, respectively, to the first, second, and third CDRs in aheavy chain variable region. As used herein, the terms “CDR-1”, “CDR-2”,and “CDR-3” refer, respectively, to the first, second and third CDRs ofeither chain's variable region.

A “framework” when used in reference to an antibody variable regionincludes all amino acid residues outside the CDR regions within thevariable region of an antibody. A variable region framework is generallya discontinuous amino acid sequence that includes only those amino acidsoutside of the CDRs. A “framework region” includes each domain of theframework that is separated by the CDRs.

A “humanized antibody” is an antibody comprising portions of antibodiesof different origin, wherein at least one portion comprises amino acidsequences of human origin. For example, the humanized antibody cancomprise portions derived from an antibody of nonhuman origin with therequisite specificity, such as a mouse, and from antibody sequences ofhuman origin (e.g., chimeric immunoglobulin), joined together chemicallyby conventional techniques (e.g., synthetic) or prepared as a contiguouspolypeptide using genetic engineering techniques (e.g., DNA encoding theprotein portions of the chimeric antibody can be expressed to produce acontiguous polypeptide chain). Another example of a humanized antibodyis an antibody containing at least one chain comprising a CDR derivedfrom an antibody of nonhuman origin and a framework region derived froma light and/or heavy chain of human origin (e.g., CDR-grafted antibodieswith or without framework changes). Chimeric or CDR-grafted single chainantibodies are also encompassed by the term humanized immunoglobulin.See, e.g., Cabilly et al., U. S. Pat. No. 4,816,567; Cabilly et al.,European Patent No. 0,125,023 B1; Boss et al., U. S. Pat. No. 4,816,397;Boss et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al.,WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276 B1;Winter, U. S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400B 1; Padlan, E. A. et al., European Patent Application No. 0,519,596 Al.See also, Ladner et al., U. S. Pat. No. 4,946,778; Huston, U. S. Pat.No. 5,476,786; and Bird, R. E. et al., Science, 242: 423-426 (1988)),regarding single chain antibodies.

In some embodiments, a humanized antibody is produced using syntheticand/or recombinant nucleic acids to prepare genes (e.g., cDNA) encodingthe desired humanized chain. For example, nucleic acid (e.g., DNA)sequences coding for humanized variable regions can be constructed usingPCR mutagenesis methods to alter DNA sequences encoding a human orhumanized chain, such as a DNA template from a previously humanizedvariable region (see e.g., Kamman, M., et al., Nucl. Acids Res., 17:5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993);Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991);and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302 (1991)). Usingthese or other suitable methods, variants can also be readily produced.For example, cloned variable regions can be mutagenized, and sequencesencoding variants with the desired specificity can be selected (e.g.,from a phage library; see e.g., Krebber et al., U. S. Pat. No.5,514,548; Hoogenboom et al., WO 93/06213, published Apr. 1, 1993).

Humanized Anti-Factor Bb Antibodies

The amino acid sequences of the mouse monoclonal anti-factor Bb antibodyfrom which the humanized anti-factor Bb antibodies described herein arederived are provided in Table 1. In some embodiments, a humanizedanti-factor Bb antibody comprises a framework region of the heavy chainvariable region and/or the light chain variable region that includesequences derived from a human immunoglobulin framework.

Table 1. Mouse Monoclonal Anti-Factor Bb Antibody

TABLE 1 Mouse Monoclonal Anti-Factor Bb Antibody Variable regions(Kabat CDRs bolded, IMGT CDRs Ab CDRs (Kabat) CDRs (IMGT) underlined)Anti-factor Bb CDR-H1: CDR-H1: V_(H) (also referred to as mouseNYAMS (SEQ ID NO: 1) GFTFSNYA (SEQ ID NO: 7) “VH0” herein): monoclonalCDR-H2: CDR-H2: EVQLVESGGALVKPG antibody TISNRGSYTYYPDSVKGISNRGSYT (SEQ ID NO: 8) GSLKLSCAASGFTFSN (SEQ ID NO: 2) YA MSWVRQTPEKRLECDR-H3: CDR-H3: WVAT ISNRGSYT YYP ERPMDY (SEQ ID NO: 3)ARERPMDY (SEQ ID NO: 9) DSVKGRFTISRDNAK NTLYLQMSSLRSEDT ALYYCARERPMDY WGQGTSVTVSS (SEQ ID NO: 12) CDR-L1: CDR-L1: V_(L) (also referred to asKASQDVGTAVA (SEQ ID QDVGTA (SEQ ID NO: 10) “Vκ0” herein): NO: 4)DIVMTQSHKFMSTSV CDR-L2: CDR-L2: GDRVSITCKAS QDVG WASTRHT (SEQ ID NO: 5)WAS (SEQ ID NO: 11) TA VAWYQQKPGQSP CDR-L3: CDR-L3: KLLIY WAS TRHTGVPHQHSSNPLT (SEQ ID NO: 6) HQHSSNPLT (SEQ ID NO: 6) DRFTGSGSGTDFTLTITNVQSEDLAVYFC HQ HSSNPLT FGAGTKLE LK (SEQ ID NO: 13)

In some embodiments, a humanized anti-factor Bb antibody describedherein comprises a heavy chain complementarity determining region 1(CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2),and a heavy chain complementarity determining region 3 (CDR-H3) of aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 12. In some embodiments, a humanized anti-factor Bb antibodydescribed herein comprises a light chain complementarity determiningregion 1 (CDR-L1), a light chain complementarity determining region 2(CDR-L2), and a light chain complementarity determining region 3(CDR-L3) of a light chain variable region comprising the amino acidsequence of SEQ ID NO: 13. In some embodiments, a humanized anti-factorBb antibody further comprises a humanized heavy chain framework regionand/or a humanized light chain framework region.

In some embodiments, according to the Kabat definition, a humanizedanti-factor Bb antibody described herein comprises a CDR-H1 comprisingthe amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acidsequence of SEQ ID NO: 3. In some embodiments, according to the Kabatdefinition, a humanized anti-factor Bb antibody described hereincomprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, aCDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR-L3comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments,a humanized anti-factor Bb antibody further comprises a humanized heavychain framework region and/or a humanized light chain framework region.In some embodiments, according to the IMGT definition, a humanizedanti-factor Bb antibody described herein comprises a CDR-H1 comprisingthe amino acid sequence of SEQ ID NO: 7, a CDR-H2 comprising the aminoacid sequence of SEQ ID NO: 8, and a CDR-H3 comprising the amino acidsequence of SEQ ID NO: 9. In some embodiments, according to the IMGTdefinition, a humanized anti-factor Bb antibody described hereincomprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10,a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and aCDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In someembodiments, a humanized anti-factor Bb antibody further comprises ahumanized heavy chain framework region and/or a humanized light chainframework region.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a V_(H) containing no more than 20 amino acidvariations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared withthe V_(H) set forth in SEQ ID NO: 12. In some embodiments, theanti-factor Bb antibody of the present disclosure comprises a V_(L)containing no more than 20 amino acid variations (e.g., no more than 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) as compared with the V_(L) set forth in SEQ ID NO: 13.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a V_(H) comprising an amino acid sequence that isat least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to theV_(H) as set forth in SEQ ID NO: 12. In some embodiments, the humanizedanti-factor Bb of the present disclosure comprises a V_(L) comprising anamino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%,or 99%) identical to the V_(L) as set forth in SEQ ID NO: 13.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising a CDR-H1 having theamino acid sequence of SEQ ID NO: 1 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 2(according to the Kabat definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 3 (according to the Kabat definitionsystem), and containing no more than 20 amino acid variations (e.g., nomore than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,3, 2, or 1 amino acid variation) in the framework regions as comparedwith the V_(H) as set forth in SEQ ID NO: 12. In some embodiments, ahumanized anti-factor Bb antibody of the present disclosure comprises ahumanized V_(L) comprising a CDR-L1 having the amino acid sequence ofSEQ ID NO: 4 (according to the Kabat definition system), a CDR-L2 havingthe amino acid sequence of SEQ ID NO: 5 (according to the Kabatdefinition system), and a CDR-L3 having the amino acid sequence of SEQID NO: 6 (according to the Kabat definition system), and containing nomore than 20 amino acid variations (e.g., no more than 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the V_(L) as setforth in SEQ ID NO: 13.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising a CDR-H1 having theamino acid sequence of SEQ ID NO: 1 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 2(according to the Kabat definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 3 (according to the Kabat definitionsystem), wherein the framework regions of the V_(H) are collectively atleast 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to theframework regions of the V_(H) as set forth in SEQ ID NO: 12. In someembodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(L) comprising a CDR-L1 having theamino acid sequence of SEQ ID NO: 4(according to the Kabat definitionsystem), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5(according to the Kabat definition system), and a CDR-L3 having theamino acid sequence of SEQ ID NO: 6 (according to the Kabat definitionsystem), wherein the framework regions of the V_(L) are collectively atleast 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the framework regions of the V_(L) as set forth in any one of SEQ ID NO: 13.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising a CDR-H1 having theamino acid sequence of SEQ ID NO: 7 (according to the IMGT definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 8(according to the IMGT definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 9 (according to the IMGT definition system),and containing no more than 20 amino acid variations (e.g., no more than20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1amino acid variation) in the framework regions as compared with theV_(H) as set forth in SEQ ID NO: 12. In some embodiments, a humanizedanti-factor Bb antibody of the present disclosure comprises a humanizedV_(L) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO:10 (according to the IMGT definition system), a CDR-L2 having the aminoacid sequence of SEQ ID NO: 11 (according to the IMGT definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6(according to the IMGT definition system), and containing no more than20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) inthe framework regions as compared with the V_(L) as set forth in SEQ IDNO: 13.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising a CDR-H1 having theamino acid sequence of SEQ ID NO: 7 (according to the IMGT definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 8(according to the IMGT definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 9 (according to the IMGT definition system),wherein the framework regions of the V_(H) collectively are at least 80%(e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the frameworkregions of the V_(H) as set forth in SEQ ID NO: 12. In some embodiments,a humanized anti-factor Bb antibody of the present disclosure comprisesa humanized V_(L) comprising a CDR-L1 having the amino acid sequence ofSEQ ID NO: 10 (according to the IMGT definition system), a CDR-L2 havingthe amino acid sequence of SEQ ID NO: 11 (according to the IMGTdefinition system), and a CDR-L3 having the amino acid sequence of SEQID NO: 6 (according to the IMGT definition system), wherein theframework region of the V_(L) collectively are at least 80% (e.g., 80%,85%, 90%, 95%, 98%, or 99%) identical to the framework regions of theV_(L) as set forth in any one of SEQ ID NO: 13.

Examples of the amino acid sequences and DNA coding sequences of thehumanized heavy chain variable regions of the humanized anti-factor Bbantibodies described herein are provided in Table 2. Examples of theamino acid sequences and DNA coding sequences of the humanized lightchain variable regions of the humanized anti-factor Bb antibodiesdescribed herein are provided in Table 3.

TABLE 2 Examples of Humanized Heavy Chain Variable Regions HumanizedV_(H) Amino Acid Sequence DNA Sequence V_(H)1 EVQLVESGGGLVKPGGSLRGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCC LSCAASGFTFSNYAMSWVRTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCA QAPGKGLEWVATISNRGSYCCTTCAGTAACTATGCCATGTCTTGGGTCCGCCAGGCTCCA TYYPDSVKGRFTISRDNAKGGGAAGGGGCTGGAGTGGGTCGCAACCATTAGTAATCGTGG NSLYLQMSSLRSEDTALYYTAGTTACACCTACTACCCAGACTCAGTGAAGGGCCGATTCA CARERPMDYWGQGTSVTVSCCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAA S (SEQ ID NO: 14)ATGAGCAGCCTGAGATCTGAGGACACGGCTTTGTATTACTGTGCGAGAGAGAGGCCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 41) V_(H)2 EVQLVESGGGLVKPGGSLRGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCC LSCAASGFTFSNYAMSWVRTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCA QAPGKGLEWVATISNRGSYCCTTCAGTAACTATGCCATGTCTTGGGTCCGCCAGGCTCCA TYYPDSVKGRFTISRDNAKGGGAAGGGGCTGGAGTGGGTCGCAACCATTAGTAATCGTGG NSLYLQMNSLRAEDTALYYTAGTTACACCTACTACCCAGACTCAGTGAAGGGCCGATTCA CARERPMDYWGQGTLVTVSCCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAA S ATGAACAGCCTGAGAGCCGAGGACACGGCTTTGTATTACTG (SEQ ID NO: 15)TGCGAGAGAGAGGCCTATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 42) V_(H)3 EVQLVESGGGLVKPGGSLRGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCC LSCAASGFTFSNYAMSWVRTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCA QAPGKGLEWVATISNRGSYCCTTCAGTAACTATGCCATGTCTTGGGTCCGCCAGGCTCCA TYYADSVKGRFTISRDNAKGGGAAGGGGCTGGAGTGGGTCGCAACCATTAGTAATCGTGG NSLYLQMNSLRAEDTALYYTAGTTACACCTACTACGCAGACTCAGTGAAGGGCCGATTCA CARERPMDYWGQGTLVTVSCCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAA S (SEQ ID NO: 16)ATGAACAGCCTGAGAGCCGAGGACACGGCTTTGTATTACTGTGCGAGAGAGAGGCCTATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 43) V_(H)4 EVQLVESGGGLVKPGGSLRGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCC LSCAASGFTFSNYAMSWVRTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCA QAPGKRLEWVATISNRGSYCCTTCAGTAACTATGCCATGTCTTGGGTCCGCCAGGCTCCA TYYPDSVKGRFTISRDNAKGGGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAATCGTGG NSLYLQMSSLRSEDTALYYTAGTTACACCTACTACCCAGACTCAGTGAAGGGCCGATTCA CARERPMDYWGQGTSVTVSCCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAA S (SEQ ID NO: 17)ATGAGCAGCCTGAGATCTGAGGACACGGCTTTGTATTACTGTGCGAGAGAGAGGCCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 44) V_(H)5 EVQLVESGGGLVKPGGSLRGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCC LSCAASGFTFSNYAMSWVRTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCA QTPEKRLEWVATISNRGSYCCTTCAGTAACTATGCCATGTCTTGGGTCCGCCAGACTCCA TYYPDSVKGRFTISRDNAKGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAATCGTGG NSLYLQMSSLRSEDTALYYTAGTTACACCTACTACCCAGACTCAGTGAAGGGCCGATTCA CARERPMDYWGQGTSVTVSCCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAA S (SEQ ID NO: 18) ATGAGCAGCCTGAGATCTGAGGACACGGCTTTGTATTACTGTGCGAGAGAGAGGCCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 45) V_(H)6 EVQLVESGGGLVKPGGSLRGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCC LSCAASGFTFSNYAMSWVRTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCA QAPGKRLEWVATISNRGSYCCTTCAGTAACTATGCCATGTCTTGGGTCCGCCAGGCTCCA TYYPDSVKGRFTISRDNAKGGGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAATCGTGG NSLYLQMNSLRAEDTALYYTAGTTACACCTACTACCCAGACTCAGTGAAGGGCCGATTCA CARERPMDYWGQGTLVTVSCCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAA S (SEQ ID NO: 19)ATGAACAGCCTGAGAGCCGAGGACACGGCTTTGTATTACTGTGCGAGAGAGAGGCCTATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 46) V_(H)7 EVQLVESGGGLVKPGGSLRGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCC LSCAASGFTFSNYAMSWVRTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCA QTPEKRLEWVATISNRGSYCCTTCAGTAACTATGCCATGTCTTGGGTCCGCCAGACTCCA TYYPDSVKGRFTISRDNAKGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAATCGTGG NSLYLQMNSLRAEDTALYYTAGTTACACCTACTACCCAGACTCAGTGAAGGGCCGATTCA CARERPMDYWGQGTLVTVSCCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAA S (SEQ ID NO: 20)ATGAACAGCCTGAGAGCCGAGGACACGGCTTTGTATTACTGTGCGAGAGAGAGGCCTATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 47) CDRs according to the Kabat definitionare bolded in the amino acid and DNA sequences

TABLE 3 Examples of humanized light chain variable regions HumanizedV_(L) (V_(κ)) Amino Acid Sequence DNA Sequence V_(κ)1DIVMTQSPSFLSASVGDRV GACATCGTGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTITCKASQDVGTAVAWYQQ TGTAGGAGACAGAGTCACCATCACTTGCAAGGCCAGTCAGGKPGQPPKLLIYWASTRHTG ATGTGGGTACTGCTGTAGCCTGGTATCAGCAAAAACCAGGGVPDRFTGSGSGTDFTLTIS CAACCTCCTAAGCTCCTGATCTATTGGGCATCCACTCGGCASLQSEDFAVYFCHQHSSNP CACTGGGGTCCCAGATAGGTTCACAGGCAGTGGATCTGGGALTFGQGTKLEIK CAGATTTCACTCTCACAATCAGCAGCCTGCAGTCTGAAGAT (SEQ ID NO: 21)TTTGCAGTTTATTTCTGTCACCAACATAGCAGCAATCCTCTCACGTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 48) V_(κ)2DIVMTQSPSTLSASVGDRV GACATCGTGATGACCCAGTCTCCATCCACCCTGTCTGCATCTITCKASQDVGTAVAWYQQ TGTAGGAGACAGAGTCACCATCACTTGCAAGGCCAGTCAGGKPGQPPKLLIYWASTRHTG ATGTGGGTACTGCTGTAGCCTGGTATCAGCAAAAACCAGGGVPDRFTGSGSGTDFTLTIS CAACCTCCTAAGCTCCTGATCTATTGGGCATCCACTCGGCASLQAEDFAVYFCHQHSSNP CACTGGGGTCCCAGATAGGTTCACAGGCAGTGGATCTGGGALTFGQGTKLEIK CAGATTTCACTCTCACAATCAGCAGCCTGCAGGCTGAAGAT (SEQ ID NO: 22)TTTGCAGTTTATTTCTGTCACCAACATAGCAGCAATCCTCTCACGTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 49) V_(κ)3DIQMTQSPSTLSASVGDRV GACATCCAGATGACCCAGTCTCCATCCACCCTGTCTGCATCTITCKASQDVGTAVAWYQQ TGTAGGAGACAGAGTCACCATCACTTGCAAGGCCAGTCAGGKPGQPPKLLIYWASTRHTG ATGTGGGTACTGCTGTAGCCTGGTATCAGCAAAAACCAGGGVPDRFSGSGSGTDFTLTIS CAACCTCCTAAGCTCCTGATCTATTGGGCATCCACTCGGCASLQAEDFAVYFCHQHSSNP CACTGGGGTCCCAGATAGGTTCAGCGGCAGTGGATCTGGGALTFGQGTKLEIK CAGATTTCACTCTCACAATCAGCAGCCTGCAGGCTGAAGAT (SEQ ID NO: 23)TTTGCAGTTTATTTCTGTCACCAACATAGCAGCAATCCTCTCACGTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 50) V_(κ)4DIQMTQSPSTLSASVGDRV GACATCCAGATGACCCAGTCTCCATCCACCCTGTCTGCATCTITCRASQDVGTAVAWYQQ TGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGGKPGQPPKLLIYWASTRHTG ATGTGGGTACTGCTGTAGCCTGGTATCAGCAAAAACCAGGGVPDRFSGSGSGTDFTLTIS CAACCTCCTAAGCTCCTGATCTATTGGGCATCCACTCGGCASLQAEDFAVYYCHQHSSNP CACTGGGGTCCCAGATAGGTTCAGCGGCAGTGGATCTGGGALTFGQGTKLEIK CAGATTTCACTCTCACAATCAGCAGCCTGCAGGCTGAAGAT (SEQ ID NO: 24)TTTGCAGTTTATTACTGTCACCAACATAGCAGCAATCCTCTCACGTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 51) V_(κ)5DIQMTQSPSTLSASVGDRV GACATCCAGATGACCCAGTCTCCATCCACCCTGTCTGCATCTITCRASQDVGTAVAWYQQ TGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGGKPGQPPKLLIYWASTRHTG ATGTGGGTACTGCTGTAGCCTGGTATCAGCAAAAACCAGGGVPDRFSGSGSGTDFTLTIS CAACCTCCTAAGCTCCTGATCTATTGGGCATCCACTCGGCASLQAEDFATYYCHQHSSNP CACTGGGGTCCCAGATAGGTTCAGCGGCAGTGGATCTGGGALTFGQGTKLEIK CAGATTTCACTCTCACAATCAGCAGCCTGCAGGCTGAAGAT (SEQ ID NO: 25)TTTGCAACTTATTACTGTCACCAACATAGCAGCAATCCTCTCACGTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 52) V_(κ)6DIVMTQSPSFLSASVGDRV GACATCGTGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTITCKASQDVGTAVAWYQQ TGTAGGAGACAGAGTCACCATCACTTGCAAGGCCAGTCAGGKPGKAPKLLIYWASTRHTG ATGTGGGTACTGCTGTAGCCTGGTATCAGCAAAAACCAGGGVPDRFTGSGSGTDFTLTIS AAAGCCCCTAAGCTCCTGATCTATTGGGCATCCACTCGGCASLQSEDFAVYFCHQHSSNP CACTGGGGTCCCAGATAGGTTCACAGGCAGTGGATCATGGGLTFGQGTKLEIK ACAGATTTCACTCTCACAATCAGCAGCCTGCAGTCTGAAGA (SEQ ID NO: 26)TTTTGCAGTTTATTTCTGTCACCAACATAGCAGCAATCCTCTCACGTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 53) V_(κ)7DIQMTQSPSTLSASVGDRV GACATCCAGATGACCCAGTCTCCATCCACCCTGTCTGCATCTITCKASQDVGTAVAWYQQ TGTAGGAGACAGAGTCACCATCACTTGCAAGGCCAGTCAGGKPGKAPKLLIYWASTRHTG ATGTGGGTACTGCTGTAGCCTGGTATCAGCAAAAACCAGGGVPDRFSGSGSGTDFTLTIS AAAGCCCCTAAGCTCCTGATCTATTGGGCATCCACTCGGCASLQAEDFAVYFCHQHSSNP CACTGGGGTCCCAGATAGGTTCAGCGGCAGTGGATCTGGGALTFGQGTKLEIK CAGATTTCACTCTCACAATCAGCAGCCTGCAGGCTGAAGAT (SEQ ID NO: 27)TTTGCAGTTTATTTCTGTCACCAACATAGCAGCAATCCTCTCACGTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 54) CDRs according to theKabat definition are bolded in the amino acid and DNA sequences

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising a CDR-H1 having theamino acid sequence of SEQ ID NO: 1 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 2(according to the Kabat definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 3 (according to the Kabat definitionsystem), and containing no more than 20 amino acid variations (e.g., nomore than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,3, 2, or 1 amino acid variation) in the framework regions as comparedwith the V_(H) as set forth in any one of SEQ ID NOs: 14-20. In someembodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(L) comprising a CDR-L1 having theamino acid sequence of SEQ ID NO: 4 (according to the Kabat definitionsystem), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5(according to the Kabat definition system), and a CDR-L3 having theamino acid sequence of SEQ ID NO: 6 (according to the Kabat definitionsystem), and containing no more than 20 amino acid variations (e.g., nomore than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,3, 2, or 1 amino acid variation) in the framework regions as comparedwith the V_(L) as set forth in any one of SEQ ID NOs: 21-27.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising a CDR-H1 having theamino acid sequence of SEQ ID NO: 1 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 2(according to the Kabat definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 3 (according to the Kabat definitionsystem), wherein the framework regions of the V_(H) are collectively atleast 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to theframework regions of the V_(H) as set forth in any one of SEQ ID NOs:14-20. In some embodiments, a humanized anti-factor Bb antibody of thepresent disclosure comprises a humanized V_(L) comprising a CDR-L1having the amino acid sequence of SEQ ID NO: 4 (according to the Kabatdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 5 (according to the Kabat definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 6 (according to the Kabatdefinition system), wherein the framework regions of the V_(L) arecollectively at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%)identical to the framework regions of the V_(L) as set forth in any oneof SEQ ID NOs: 21-27.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising a CDR-H1 having theamino acid sequence of SEQ ID NO: 7 (according to the IMGT definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 8(according to the IMGT definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 9 (according to the IMGT definition system),and containing no more than 20 amino acid variations (e.g., no more than20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1amino acid variation) in the framework region as compared with the V_(H)as set forth in any one of SEQ ID NOs: 14-20. In some embodiments, ahumanized anti-factor Bb antibody of the present disclosure comprises ahumanized V_(L) comprising a CDR-L1 having the amino acid sequence ofSEQ ID NO: 10 (according to the IMGT definition system), a CDR-L2 havingthe amino acid sequence of SEQ ID NO: 11 (according to the IMGTdefinition system), and a CDR-L3 having the amino acid sequence of SEQID NO: 6 (according to the IMGT definition system), and containing nomore than 20 amino acid variations (e.g., no more than 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework region as compared with the V_(L) as setforth in any one of SEQ ID NOs: 21-27.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising a CDR-H1 having theamino acid sequence of SEQ ID

NO: 7 (according to the IMGT definition system), a CDR-H2 having theamino acid sequence of SEQ ID NO: 8 (according to the IMGT definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 9(according to the IMGT definition system), wherein the framework regionof the V_(H) is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%)identical to the framework regions of the V_(H) as set forth in any oneof SEQ ID NOs: 14-20. In some embodiments, a humanized anti-factor Bbantibody of the present disclosure comprises a humanized V_(L)comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 10(according to the IMGT definition system), a CDR-L2 having the aminoacid sequence of SEQ ID NO: 11 (according to the IMGT definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6(according to the IMGT definition system), wherein the framework regionof the V_(L) are collectively at least 80% (e.g., 80%, 85%, 90%, 95%,98%, or 99%) identical to the framework regions of the V_(L) as setforth in any one of SEQ ID NOs: 21-27.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) as set forth in any one of SEQ IDNOs: 14-20. In some embodiments, a humanized anti-factor Bb antibody ofthe present disclosure comprises a humanized V_(L) as set forth in anyone of SEQ ID NOs: 21-27. Table 4 provides examples of humanizedanti-factor Bb antibodies comprising one of the humanized V_(H) sprovided in Table 2 and one of the humanized V_(L)s provided in Table 3.

TABLE 4Examples of Variable Regions of Humanized Anti-factor Bb AntibodiesAntibody Variable Region Amino Acid Sequence V_(H)6/V_(κ)7 V_(H):EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKRLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARERPMDYWGQGTLVTVSS(SEQ ID NO: 19) V_(L):DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDFAVYFCHQHSSNPLTFGQGTKLEIK (SEQ ID NO: 27)V_(H)4/V_(κ)6 V_(H):EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKRLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMSSLRSEDTALYYCARERPMDYWGQGTSVTVSS(SEQ ID NO: 17) V_(L):DIVMTQSPSFLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISSLQSEDFAVYFCHQHSSNPLTFGQGTKLEIK (SEQ ID NO: 26)V_(H)6/V_(κ)6 V_(H):EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKRLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARERPMDYWGQGTLVTVSS(SEQ ID NO: 19) V_(L):DIVMTQSPSFLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISSLQSEDFAVYFCHQHSSNPLTFGQGTKLEIK (SEQ ID NO: 26)V_(H)4/V_(κ)7 V_(H):EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKRLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMSSLRSEDTALYYCARERPMDYWGQGTSVTVSS(SEQ ID NO: 17) V_(L):DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDFAVYFCHQHSSNPLTFGQGTKLEIK (SEQ ID NO: 27)V_(H)7/V_(κ)K7 V_(H):EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWVRQTPEKRLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARERPMDYWGQGTLVTVSS(SEQ ID NO: 20) V_(L):DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDFAVYFCHQHSSNPLTFGQGTKLEIK (SEQ ID NO: 27)V_(H)1/V_(κ)1 V_(H):EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMSSLRSEDTALYYCARERPMDYWGQGTSVTVSS(SEQ ID NO: 14) V_(L):DIVMTQSPSFLSASVGDRVTITCKASQDVGTAVAWYQQKPGQPPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISSLQSEDFAVYFCHQHSSNPLTFGQGTKLEIK (SEQ ID NO: 21)V_(H)1/V_(κ)2 V_(H):EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMSSLRSEDTALYYCARERPMDYWGQGTSVTVSS(SEQ ID NO: 14) V_(L):DIVMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGQPPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISSLQAEDFAVYFCHQHSSNPLTFGQGTKLEIK (SEQ ID NO: 22)V_(H)1/V_(κ)3 V_(H):EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMSSLRSEDTALYYCARERPMDYWGQGTSVTVSS(SEQ ID NO: 14) V_(L):DIQMTQSPSTLSASVGDRVTITCKASQDVGTAVAWYQQKPGQPPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDFAVYFCHQHSSNPLTFGQGTKLEIK (SEQ ID NO: 23)V_(H)1/V_(κ)4 V_(H):EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMSSLRSEDTALYYCARERPMDYWGQGTSVTVSS(SEQ ID NO: 14) V_(L):DIQMTQSPSTLSASVGDRVTITCRASQDVGTAVAWYQQKPGQPPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDFAVYYCHQHSSNPLTFGQGTKLEIK (SEQ ID NO: 24)V_(H)1/V_(κ)5 V_(H):EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVATISNRGSYTYYPDSVKGRFTISRDNAKNSLYLQMSSLRSEDTALYYCARERPMDYWGQGTSVTVSS(SEQ ID NO: 14) V_(L):DIQMTQSPSTLSASVGDRVTITCRASQDVGTAVAWYQQKPGQPPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDFATYYCHQHSSNPLTFGQGTKLEIK (SEQ ID NO: 25) CDRsaccording to the Kabat definition are bolded in the amino acid and DNAsequences

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising the amino acidsequence of SEQ ID NO: 19 and a humanized V_(L) comprising the aminoacid sequence of SEQ ID NO: 27.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising the amino acidsequence of SEQ ID NO: 17 and a humanized V_(L) comprising the aminoacid sequence of SEQ ID NO: 26.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising the amino acidsequence of SEQ ID NO: 19 and a humanized V_(L) comprising the aminoacid sequence of SEQ ID NO: 26.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising the amino acidsequence of SEQ ID NO: 17 and a humanized V_(L) comprising the aminoacid sequence of SEQ ID NO: 27.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising the amino acidsequence of SEQ ID NO: 20 and a humanized V_(L) comprising the aminoacid sequence of SEQ ID NO: 27.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising the amino acidsequence of SEQ ID NO: 14 and a humanized V_(L) comprising the aminoacid sequence of any one of SEQ ID NOs: 21.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising the amino acidsequence of SEQ ID NO: 14 and a humanized V_(L) comprising the aminoacid sequence of any one of SEQ ID NOs: 22.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising the amino acidsequence of SEQ ID NO: 14 and a humanized V_(L) comprising the aminoacid sequence of any one of SEQ ID NOs: 23.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising the amino acidsequence of SEQ ID NO: 14 and a humanized V_(L) comprising the aminoacid sequence of any one of SEQ ID NOs: 24.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure comprises a humanized V_(H) comprising the amino acidsequence of SEQ ID NO: 14 and a humanized V_(L) comprising the aminoacid sequence of any one of SEQ ID NOs: 25.

In some embodiments, a humanized anti-factor Bb antibody describedherein is a full-length IgG, an Ig monomer, a Fab fragment, a F(ab′)₂fragment, a scFv, a scAb, or a Fv. In some embodiments, a humanizedanti-factor Bb antibody described herein is a full-length IgG. In someembodiments, the heavy chain of any of the humanized anti-factor Bbantibodies as described herein comprises a heavy chain constant region(CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combinationthereof). The heavy chain constant region can of any suitable origin,e.g., human, mouse, rat, or rabbit. In some embodiments, the heavy chainconstant region is from a human IgG (a gamma heavy chain), e.g., IgG1,IgG2, or IgG4.

In some embodiments, mutations can be introduced into the heavy chainconstant region of any one of the humanized anti-factor Bb antibodiesdescribed herein. In some embodiments, one, two or more mutations (e.g.,amino acid substitutions) are introduced into the heavy chain constantregion (e.g., in a CH2 domain (residues 231-340 of human IgG1) and/orCH3 domain (residues 341-447 of human IgG1) and/or the hinge region,with numbering according to the Kabat numbering system (e.g., the EUindex in Kabat)) to increase or decrease the affinity of the antibodyfor an Fc receptor (e.g., an activated Fc receptor) on the surface of aneffector cell. Mutations in the Fc region of an antibody that decreaseor increase the affinity of an antibody for an Fc receptor andtechniques for introducing such mutations into the Fc receptor orfragment thereof are known to one of skill in the art. Examples ofmutations in the Fc receptor of an antibody that can be made to alterthe affinity of the antibody for an Fc receptor are described in, e.g.,Smith P et al., (2012) PNAS 109: 6181-6186, U. S. Pat. No. 6,737,056,and International Publication Nos. WO 02/060919; WO 98/23289; and WO97/34631, which are incorporated herein by reference. In someembodiments, one, two or more mutations (e.g., amino acid substitutions)are introduced into the hinge region of the heavy chain constant region(CH1 domain) such that the number of cysteine residues in the hingeregion are altered (e.g., increased or decreased) as described in, e.g.,U. S. Pat. No. 5,677,425. The number of cysteine residues in the hingeregion of the CH1 domain can be altered to, e.g., facilitate assembly ofthe light and heavy chains, or to alter (e.g., increase or decrease) thestability of the antibody or to facilitate linker conjugation.

In some embodiments, one, two or more amino acid mutations (i.e.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof to alter (e.g.,decrease or increase) half-life of the antibody in vivo. In someembodiments, the one or more mutations are introduced into an Fc orhinge-Fc domain fragment. See, e.g., International Publication Nos. WO02/060919; WO 98/23289; and WO 97/34631; and U. S. Pat. Nos. 5,869,046;6,121,022; 6,277,375; and 6,165,745 for examples of mutations that willalter (e.g., decrease or increase) the half-life of an antibody in vivo.

In some embodiments, the constant region antibody described herein is anIgG1 constant region and comprises a methionine (M) to tyrosine (Y)substitution in position 252, a serine (S) to threonine (T) substitutionin position 254, and a threonine (T) to glutamic acid (E) substitutionin position 256, numbered according to the EU index as in Kabat. See U.S. Pat. No. 7,658,921, which is incorporated herein by reference. Thistype of mutant IgG, referred to as “YTE mutant” has been shown todisplay fourfold increased half-life as compared to wild-type versionsof the same antibody (see Dall'Acqua W F et al., (2006) J Biol Chem 281:23514-24). In some embodiments, an antibody comprises an IgG constantdomain comprising one, two, three or more amino acid substitutions ofamino acid residues at positions 251-257, 285-290, 308-314, 385-389, and428-436, numbered according to the EU index as in Kabat. Additionalmutations that may be introduced to the heavy chain constant region thatwould increase the half-life of the antibody are known in the art, e.g.,the M428L/N434S (EU numbering; M459L/N466S Kabat numbering) mutations asdescribed in Zalevsky et al., Nat Biotechnol. 2010 February; 28(2):157-159.

In some embodiments, one, two or more amino acid substitutions areintroduced into an IgG constant domain Fc region to alter the effectorfunction(s) of the antibody. The effector ligand to which affinity isaltered can be, for example, an Fc receptor or the Cl component ofcomplement. This approach is described in further detail in U. S. Pat.Nos. 5,624,821 and 5,648,260. In some embodiments, the deletion orinactivation (through point mutations or other means) of a constantregion domain can reduce Fc receptor binding of the circulating antibodythereby increasing tumor localization. See, e.g., U. S. Pat. Nos.5,585,097 and 8,591,886 for a description of mutations that delete orinactivate the constant domain and thereby increase tumor localization.In some embodiments, at least one amino acid substitutions may beintroduced into the Fc region of an antibody described herein to removepotential glycosylation sites on Fc region, which may reduce Fc receptorbinding (see, e.g., Shields R L et al., (2001) J Biol Chem 276:6591-604).

In some embodiments, at least one amino acid in the constant region canbe replaced with a different amino acid residue such that the antibodyhas altered Clq binding and/or reduced or abolished complement dependentcytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 (Idusogie et al.). In some embodiments, at leastone amino acid residue in the N-terminal region of the CH2 domain of anantibody described herein is altered to thereby alter the ability of theantibody to fix complement. This approach is described further inInternational Publication No. WO 94/29351. In some embodiments, the Fcregion of an antibody described herein is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcy receptor. This approach is described further in InternationalPublication No. WO 00/42072.

In some embodiments, to avoid potential complications due to Fab-armexchange, which is known to occur with native IgG4 mAbs, the antibodiesprovided herein may comprise a stabilizing ‘Adair’ mutation (Angal S.,et al., “A single amino acid substitution abolishes the heterogeneity ofchimeric mouse/human (IgG4) antibody,” Mol Immunol 30, 105-108; 1993),where serine 228 (EU numbering; residue 241 Kabat numbering) isconverted to proline resulting in an IgG1-like hinge sequence. In someembodiments, to reduce residual antibody-dependent cellularcytotoxicity, a L235E (EU numbering, corresponding to L248E in Kabatnumbering) mutation is introduced to the heavy chain constant region,e.g., as described in Benhnia et al., JOURNAL OF VIROLOGY, Dec. 2009, p.12355-12367.

In some embodiments, the heavy chain constant region in any one of thehumanized anti-factor Bb antibodies described herein is an IgG4 constantregion, or a variant there of. Examples of IgG4 constant regions andvariants are provided in Table 5.

TABLE 5 Examples of Heavy Chain Constant Regions Heavy ChainConstant Region Amino Acid Sequence IgG4 constantASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL region WTQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEF (also referredLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP to herein asREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV “IgG4wt”)YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 28) IgG4 constantASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLregion variant 1QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF (also referredEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP to herein asREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV “IgG4v1”)YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 29) IgG4 constantASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLregion variant 2QSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF (also referredEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP to herein asREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV “IgG4v2”)YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK (SEQ ID NO: 30)

In some embodiments, the light chain of any of the humanized anti-factorBb antibodies described herein may further comprise a light chainconstant region (C_(L)). In some examples, the C_(L) is a kappa lightchain. In other examples, the C_(L) is a lambda light chain. In someembodiments, the C_(L) is a kappa light chain, the sequence of which isprovided below:

(SEQ ID NO: 31) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC

Other antibody heavy and light chain constant regions are well known inthe art, e.g., those provided in the IMGT database (www.imgt.org) or atwww.vbase2.org/vbstat.php., both of which are incorporated by referenceherein.

In some embodiments, a humanized anti-factor Bb antibody describedherein comprises a heavy chain comprising any one of the V_(H) as listedin Table 2 or any variants thereof and a heavy chain constant regionthat is at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical to any one of SEQ ID NOs: 28-30. In someembodiments, a humanized anti-factor Bb antibody described hereincomprises a heavy chain comprising any one of the V_(H) as listed inTable 2 or any variants thereof and a heavy chain constant region thatcontains no more than 20 amino acid variations (e.g., no more than 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1amino acid variation) as compared with any one of SEQ ID NOs: 28-30. Insome embodiments, a humanized anti-factor Bb antibody described hereincomprises a heavy chain comprising any one of the V_(H) as listed inTable 2 or any variants thereof and a heavy chain constant regioncomprising the amino acid sequence of any one of SEQ ID NOs: 28-30.

In some embodiments, a humanized anti-factor Bb antibody describedherein comprises a light chain comprising any one of the V_(L) as listedin Table 3 or any variants thereof and a light chain constant regionthat is at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical to SEQ ID NO: 31. In some embodiments, a humanizedanti-factor Bb antibody described herein comprises a light chaincomprising any one of the V_(L) as listed in Table 3 or any variantsthereof and a light chain constant region that contains no more than 20amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) ascompared with SEQ ID NO: 31. In some embodiments, a humanizedanti-factor Bb antibody described herein comprises a light chaincomprising any one of the V_(L) as listed in Table 3 or any variantsthereof and a light chain constant region comprising the amino acidsequence of SEQ ID NO: 31.

Examples of the amino acid sequences of the heavy chain and light chainof the humanized anti-factor Bb antibodies described herein are providedin Table 6.

TABLE 6 Examples of the heavy chain and light chainof the humanized anti-factor Bb antibodies Antibody Amino Acid SequenceV_(H)6/V_(κ)7- Heavy chain IgG4wt EVQLVESGGGLVKPGGSLRLSCAASGFTFS

WVRQAPGKRLEWVA

RFTISRDNAKNSLYLQMNSLRAEDTALYYCAR

WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 32) Light Chain DIQMTQSPSTLSASVGDRVTITC

WYQQKPGKAPKLLIY

GVPDRF SGSGSGTDFTLTISSLQAEDFAVYFC

FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 35) V_(H)6/V_(κ)7-EVQLVESGGGLVKPGGSLRLSCAASGFTFS

WVRQAPGKRLEWVA

IgG4v1

RFTISRDNAKNSLYLQMNSLRAEDTALYYCAR

WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 33) Light Chain DIQMTQSPSTLSASVGDRVTITC

WYQQKPGKAPKLLIY

GVPDRFS GSGSGTDFTLTISSLQAEDFAVYFC

FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 35) V_(H)6/V_(κ)7-EVQLVESGGGLVKPGGSLRLSCAASGFTFS

WVRQAPGKRLEWVA

IgG4v2

RFTISRDNAKNSLYLQMNSLRAEDTALYYCAR

WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK (SEQ ID NO: 34) Light Chain DIQMTQSPSTLSASVGDRVTITC

WYQQKPGKAPKLLIY

GVPDRF SGSGSGTDFTLTISSLQAEDFAVYFC

FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 35) V_(H)4/V_(κ)6- Heavy ChainIgG4wt EVQLVESGGGLVKPGGSLRLSCAASGFTFS

WVRQAPGKRLEWVA

RFTISRDNAKNSLYLQMSSLRSEDTALYYCA

WGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 36) Light Chain DIVMTQSPSFLSASVGDRVTITC

WYQQKPGKAPKLLIY

GVPDRF TGSGSGTDFTLTISSLQSEDFAVYFC

FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 39) V_(H)4/V_(κ)6- Heavy ChainIgG4v1 EVQLVESGGGLVKPGGSLRLSCAASGFTFS

WVRQAPGKRLEWVA

RFTISRDNAKNSLYLQMSSLRSEDTALYYCAR

WGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 37) Light Chain DIVMTQSPSFLSASVGDRVTITC

WYQQKPGKAPKLLIY

GVPDRF TGSGSGTDFTLTISSLQSEDFAVYFC

FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 39) V_(H)4/V_(κ)6- Heavy ChainIgG4v2 EVQLVESGGGLVKPGGSLRLSCAASGFTFS

WVRQAPGKRLEWVA

RFTISRDNAKNSLYLQMSSLRSEDTALYYCAR

WGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLGK (SEQ ID NO: 38) Light Chain DIVMTQSPSFLSASVGDRVTITC

WYQQKPGKAPKWY

GVPDRFTG SGSGTDFTLTISSLQSEDFAVYFC

FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 39)

In some embodiments, a humanized anti-factor Bb antibody comprises aheavy chain comprising the amino acid sequences of SEQ ID NO: 32 and alight chain comprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments, a humanized anti-factor Bb antibody comprises aheavy chain comprising the amino acid sequences of SEQ ID NO: 33 and alight chain comprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments, a humanized anti-factor Bb antibody comprises aheavy chain comprising the amino acid sequences of SEQ ID NO: 34 and alight chain comprising the amino acid sequence of SEQ ID NO: 35.

In some embodiments, a humanized anti-factor Bb antibody comprises aheavy chain comprising the amino acid sequences of SEQ ID NO: 36 and alight chain comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, a humanized anti-factor Bb antibody comprises aheavy chain comprising the amino acid sequences of SEQ ID NO: 37 and alight chain comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, a humanized anti-factor Bb antibody comprises aheavy chain comprising the amino acid sequences of SEQ ID NO: 38 and alight chain comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure binds a factor Bb protein (e.g., a factor Bb protein from amammal, fish, or invertebrate that has a complement system). In someembodiments, a humanized anti-factor Bb antibody of the presentdisclosure binds a mammalian factor Bb protein. In some embodiments, ahumanized anti-factor Bb antibody of the present disclosure binds ahuman factor Bb protein. In some embodiments, a humanized anti-factor Bbantibody of the present disclosure binds a factor Bb protein having theamino acid sequence of SEQ ID NO: 40.

Homo sapiens factor Bb protein (SEQ ID NO: 40)KIVLDPSGSMNIYLVLDGSDSIGASNFTGAKKCLVNLIEKVASYGVKPRYGLVTYATYPKIWVKVSEADSSNADWVTKQLNEINYEDHKLKSGTNTKKALQAVYSMMSWPDDVPPEGWNRTRHVIILMTDGLHNMGGDPITVIDEIRDLLYIGKDRKNPREDYLDVYVFGVGPLVNQVNINALASKKDNEQHVFKVKDMENLEDVFYQMIDESQSLSLCGMVWEHRKGTDYHKQPWQAKISVIRPSKGHESCMGAVVSEYFVLTAAHCFTVDDKEHSIKVSVGGEKRDLEIEVVLFHPNYNINGKKEAGIPEFYDYDVALIKLKNKLKYGQTIRPICLPCTEGTTRALRLPPTTTCQQQKEELLPAQDIKALFVSEEEKKLTRKEVYIKNGDKKGSCERDAQYAPGYDKVKDISEVVTPRFLCTGGVSPYADPNTCRGDSGGPLIVHKRSRFIQVGVISWGVVDVCKNQKRQKQVPAHARDFHINLFQVLPWLKEKLQDE DLGFL

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure binds a complement Bb protein with an affinity of about 10⁻⁶to 10⁻¹¹ nM, e.g., from about 10⁻⁶ M to about 10⁻⁷ M, about 10⁻⁷ M toabout 10⁻⁸ M, about 10⁻⁸ M to about 10⁻⁹ M, from about 10⁻⁹ M to about10⁻¹⁰M, or from about 10⁻¹⁰ M to about 10⁻¹¹ M. The terms “about”preceding a numerical value mean ±10% of the recited numerical value.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure exhibits preferential binding for factor Bb, compared withbinding for factor B. In some embodiments, a humanized anti-factor Bbantibody of the present disclosure binds to factor Bb, but does notsubstantially bind to soluble Factor B. In some embodiments, a humanizedanti-factor Bb antibody of the present disclosure binds to factor Bbwith an affinity that is at least 2-fold, at least 2.5-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 7.5-fold, at least10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least50-fold, at least 75-fold, or at least 100-fold, higher than theaffinity of the antibody for factor B. In some embodiments, a humanizedanti-factor Bb antibody of the present disclosure binds to factor Bbwith an affinity that is 2-fold to 2.5-fold, 2.5-fold to 5-fold, 5-foldto 10-fold, 10-fold to 15-fold, 15-fold to 20-fold, 20-fold to 25-fold,25-fold to 50-fold, 50-fold to 75-fold, or 75-fold to 100-fold, higherthan the affinity of the antibody for factor B.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure inhibits the complement pathway activity by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or 100%, compared to the level of complementactivity in the absence of a humanized anti-factor Bb antibody.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure inhibits alternative pathway (AP) activity by at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or 100%, compared to the level of AP activityin the absence of a humanized anti-factor Bb antibody. In someembodiments, a humanized anti-factor Bb antibody of the presentdisclosure inhibits AP activity with an IC₅₀ of 10⁻⁷ M to 10⁻⁹M, e.g.,an IC₅₀ of 10⁻⁷ M to 5×10⁻⁷ M, 5×10⁻⁷ M to 10⁻⁸M, 10⁻⁸M to 5×10⁻⁸ M, or5×10⁻⁸ M to 10⁻⁹M. In some embodiments, the complement AP activity isselected the group consisting of AP-mediated terminal membrane attackcomplex (MAC) deposition, AP-mediated hemolysis, C3 fragment depositionon red blood cells or other cell types, C3b/Bb-mediated cleavage of C3,and C3bBb3b-mediated cleavage of C5. In some embodiments, inhibition ofcomplement AP activity by the humanized anti-factor Bb antibodies can bemeasured using the Complement System Alternative Pathway WIESLAB® kit.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure inhibits formation of membrane attack complex (MAC) by atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or 100%, compared to the amountof MAC formed in the absence of the anti-factor Bb antibody.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure inhibits C3b/Bb-mediated cleavage of C3. C3b/Bb is also knownas “C3 convertase.” In some embodiments, a humanized anti-factor Bbantibody of the present disclosure inhibits C3b/Bb-mediated cleavage ofC3 by at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or 100%, compared to thecleavage of C3 in the absence of the anti-factor Bb antibody. In someembodiments, a humanized anti-factor Bb antibody of the presentdisclosure inhibits

C3b/Bb-mediated cleavage of C3 with an IC₅₀ of from 10⁻⁷ M to 10⁻⁹ M,e.g., an IC₅₀ of from 10⁻⁷ M to 5×10⁻⁷ M, from 5×10⁻⁷ M to 10⁻⁸M, from10⁻⁸M to 5×10⁻⁸M, or from 5×10⁻⁸ M to 10⁻⁹ M.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure inhibits C3b/Bb-mediated cleavage of C3, thereby reducingproduction of a C3 cleavage product. For example, a humanizedanti-factor Bb antibody of the present disclosure may inhibitC3b/Bb-mediated cleavage of C3, thereby reducing production of a C3cleavage product (e.g., C3a and/or C3b) by at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or 100%, compared to the production of the C3 cleavageproduct in the absence of the anti-factor Bb antibody.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure inhibits complement AP-mediated cell lysis by at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or 100%, compared to the degree of cell lysisin the absence of the anti-factor Bb antibody. A cell lysis assay can beused to determine the degree of inhibition of AP-mediated cell lysis. Insome embodiments, a humanized anti-factor Bb antibody of the presentdisclosure inhibits AP-mediated cell lysis with an IC₅₀ of from 10⁻⁷ Mto 10⁻⁹M, e.g., an IC₅₀ of from 10⁻⁷ M to 5×10⁻⁷ M, from 5×10⁻⁷ M to10⁻⁸ M, from 10⁻⁸M to 5×10⁻⁸ M, or from 5×10⁻⁸ M to 10⁻⁹M.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure inhibits complement AP-mediated hemolysis by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or 100%, compared to the degree of hemolysis inthe absence of the anti-factor Bb antibody. A rabbit red blood cell(RBC) hemolysis assay can be used to determine the degree of inhibitionof AP-mediated hemolysis. In some embodiments, a humanized anti-factorBb antibody of the present disclosure inhibits AP-mediated hemolysiswith an IC₅₀ of from 10⁻⁷ M to 10⁻⁹M, e.g., an IC₅₀ of from 10⁻⁷ M to5×10⁻⁷ M, from 5×10⁻⁷ M to 10⁻⁸ M, from 10⁻⁸ M to 5×10⁻⁸ M, or from5×10⁻⁸ M to 10⁻⁹M. In some embodiments, a humanized anti-factor Bbantibody of the present disclosure inhibits AP-mediated deposition ofC3b, C3d, or other C3 split product on a cell or tissue. For example, ahumanized anti-factor Bb antibody of the present disclosure may inhibitAP-mediated deposition of C3b, C3d, or other C3 split product on a cellor tissue by at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or 100%, comparedto the amount of deposition of C3b, C3d, or other C3 split product onthe cell or tissue in the absence of administration of the anti-factorBb antibody, or before administration of the anti-factor Bb antibody. Insome embodiments, a humanized anti-factor Bb antibody of the presentdisclosure inhibits AP-mediated deposition of C3b, C3d, or other C3split product on a cell or tissue with an IC₅₀ of from 10⁻⁷ M to 10⁻⁹ M,e.g., an IC₅₀ of from 10⁻⁷ M to 5×10⁻⁷ M, from 5×10⁻⁷ M to 10⁻⁸ M, from10⁻⁸ M to 5×10⁻⁸ M, or from 5×10⁻⁸ M to 10⁻⁹M.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure inhibits AP-mediated C3b deposition on a cell or tissue by atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or 100%, compared to the amountof C3b deposition on the cell or tissue in the absence of the humanizedanti-factor Bb antibody. In some embodiments, a humanized anti-factor Bbantibody of the present disclosure inhibits AP-mediated C3b depositionon a cell or tissue with an IC₅₀ of from 10⁻⁷ M to 10⁻⁹M, e.g., an IC₅₀of from 10⁻⁷ M to 5×10⁻⁷ M, from 5×10⁻⁷ M to 10⁻⁸ M, from 10⁻⁸ M to5×10⁻⁸ M, or from 5×10⁻⁸ M to 10⁻⁹M.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure inhibits AP-mediated C3b deposition on red blood cells (RBCs)or other cell types by at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or100%, compared to the amount of C3b deposition on RBCs in the absence ofthe humanized anti-factor Bb antibody. In some embodiments, a humanizedanti-factor Bb antibody of the present disclosure inhibits AP-mediatedC3b deposition on RBCs with an IC₅₀ of from 10⁻⁷ M to 10⁻⁹ M, e.g., anIC₅₀ of from 10⁻⁷ M to 5×10⁻⁷ M, from 5×10⁻⁷ M to 10⁻⁸ M, from 10⁻⁸ M to5×10⁻⁸ M, or from 5×10⁻⁸ M to 10⁻⁹ M.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure, when administered to a subject in need thereof, reduces theamount of factor Bb in circulation in the subject. For example, ahumanized anti-factor Bb antibody of the present disclosure, whenadministered to a subject in need thereof, may reduce the amount offactor Bb in circulation in the subject by at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95%, compared to the amount of factor Bb in circulation in thesubject in the absence of administering the humanized anti-factor Bbantibody, or compared to the amount of factor Bb in circulation in thesubject before administration of the humanized anti-factor Bb antibody.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure inhibits C3bBb3b-mediated cleavage of C5. In someembodiments, a humanized anti-factor Bb antibody of the presentdisclosure inhibits C3bBb3b-mediated cleavage of C5 by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95%, compared to C3bBb3b-mediated cleavage of C5in the absence of a humanized anti-factor Bb antibody.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure is a bispecific or multispecific antibody. For example, ahumanized anti-factor Bb antibody can be a bispecific antibodycomprising a first antigen-binding portion that specifically binds anepitope in a complement Bb protein, and a second antigen-binding portionthat binds a second antigen.

Immunoconjugates

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure is conjugated with another agent to form an immunoconjugate.For example, a humanized anti-factor Bb antibody may comprise a freethiol (-SH) group at the carboxyl terminus, where the free thiol groupcan be used to attach the antibody to a second polypeptide (e.g.,another antibody, including a humanized anti-factor Bb antibody), ascaffold, a carrier, etc. In some embodiments, a humanized anti-factorBb antibody comprises at least one non-naturally occurring amino acids.In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group, an acetyl group, an aminooxy group, a hydrazine group, ahydrazide group, a semicarbazide group, an azide group, or an alkynegroup. See, e.g., U. S. Pat. No. 7,632,924 for suitable non-naturallyoccurring amino acids. Inclusion of a non-naturally occurring amino acidcan provide for linkage to a polymer, a second polypeptide, a scaffold,etc. For example, a humanized anti-factor Bb antibody linked to awater-soluble polymer can be made by reacting a water-soluble polymer(e.g., PEG) that comprises a carbonyl group to the antibody, where theantibody comprises a non-naturally encoded amino acid that comprises anaminooxy, hydrazine, hydrazide or semicarbazide group. In someembodiments, a humanized anti-factor Bb antibody linked to awater-soluble polymer can be made by reacting the antibody thatcomprises an alkyne-containing amino acid with a water-soluble polymer(e.g., PEG) that comprises an azide moiety. In some embodiments, theazide or alkyne group is linked to the PEG molecule through an amidelinkage.

In some embodiments, a humanized anti-factor Bb antibody is linked(e.g., covalently linked) to a polymer (e.g., a polymer other than apolypeptide). Suitable polymers include, e.g., biocompatible polymers,and water-soluble biocompatible polymers. Suitable polymers includesynthetic polymers and naturally-occurring polymers. Suitable polymerscan have an average molecular weight in a range of from 500 Da to 50,000Da, e.g., from 5,000 Da to 40,000 Da, or from 25,000 to 40,000 Da.

In some embodiments, a humanized anti-factor Bb antibody comprises a“radiopaque” label, e.g., a label that can be easily visualized usingfor example x-rays. Radiopaque materials are well known to those ofskill in the art. The most common radiopaque materials include iodide,bromide or barium salts. Other radiopaque materials are also known andinclude but are not limited to organic bismuth derivatives (see, e.g.,U. S. Pat. No. 5,939,045), radiopaque multiurethanes (see U. S. Pat. No.5,346,981), organobismuth composites (see, e.g., U. S. Pat. No.5,256,334), and/or radiopaque barium multimer complexes (see, e.g., U.S. Pat. No. 4,866,132).

In some embodiments, a humanized anti-factor Bb antibody is covalentlylinked to a second moiety (e.g., a lipid, a polypeptide other than ahumanized anti-factor Bb antibody, a synthetic polymer, and/or acarbohydrate) using for example, glutaraldehyde, a homobifunctionalcross-linker, or a heterobifunctional cross-linker.

In some embodiments, a humanized anti-factor Bb antibody is immobilizedon a solid support. Suitable supports are well known in the art andcomprise, inter alia, commercially available column materials,polystyrene beads, latex beads, magnetic beads, colloid metal particles,glass and/or silicon chips and surfaces, nitrocellulose strips, nylonmembranes, sheets, duracytes, wells of reaction trays (e.g., multi-wellplates), plastic tubes, etc. A solid support can comprise any of avariety of substances, including, e.g., glass, polystyrene, polyvinylchloride, polypropylene, polyethylene, polycarbonate, dextran, nylon,amylose, natural and modified celluloses, polyacrylamides, agaroses, andmagnetite. Suitable methods for immobilizing a humanized anti-factor Bbantibody onto a solid support are well known and include, but are notlimited to ionic, hydrophobic, and/or covalent interactions. Solidsupports can be soluble or insoluble, e.g., in aqueous solution. In someembodiments, a suitable solid support is generally insoluble in anaqueous solution.

In some embodiments, a humanized anti-factor Bb antibody comprises adetectable label. Suitable detectable labels include any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means. Suitable include, but are notlimited to, magnetic beads (e.g. DYNABEADS℠), fluorescent dyes (e.g.,fluorescein isothiocyanate, Texas Red, rhodamine, a green fluorescentprotein, a red fluorescent protein, and/or a yellow fluorescentprotein), radiolabels (e.g., ³H ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase, luciferase, and otherscommonly used in an enzyme-linked immunosorbent assay (ELISA)), andcolorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads.

In some embodiments, a humanized anti-factor Bb antibody comprises acontrast agent or a radioisotope, where the contrast agent orradioisotope is one that is suitable for use in imaging, e.g., imagingprocedures carried out on humans. Non-limiting examples of labelsinclude radioisotope such as ¹²³I (iodine), ¹⁸F (fluorine), ⁹⁹Tc(technetium), ¹¹¹In (indium), and ⁶⁷Ga (gallium), and contrast agentsuch as gadolinium (Gd), dysprosium, and iron. Radioactive Gd isotopes(¹⁵³Gd) also are available and suitable for imaging procedures innon-human mammals. A humanized anti-factor Bb antibody can be labeledusing standard techniques. For example, a humanized anti-factor Bbantibody can be iodinated using chloramine T or1,3,4,6-tetrachloro-3α,6α-diphenylglycouril. For fluorination, fluorineis added to a humanized anti-factor Bb antibody during the synthesis bya fluoride ion displacement reaction. See, Muller-Gartner, H., TIBTech., 16:122-130 (1998) and Saji, H., Crit. Rev. Ther. Drug CarrierSyst., 16(2):209-244 (1999) for a review of synthesis of proteins withsuch radioisotopes. A humanized anti-factor Bb antibody can also belabeled with a contrast agent through standard techniques. For example,a humanized anti-factor Bb antibody can be labeled with Gd byconjugating low molecular Gd chelates such as Gd diethylene triaminepentaacetic acid (GdDTPA) or Gd tetraazacyclododecanetetraacetic(GdDOTA) to the antibody. See, Caravan et al., Chem. Rev. 99:2293-2352(1999) and Lauffer et al., J. Magn. Reson. Imaging, 3:11-16 (1985). Ahumanized anti-factor Bb antibody can be labeled with Gd by, forexample, conjugating polylysine-Gd chelates to the antibody. See, forexample, Curtet et al., Invest. Radiol., 33(10):752-761 (1998).Alternatively, in some embodiments, a humanized anti-factor Bb antibodycan be labeled with Gd by incubating paramagnetic polymerized liposomesthat include Gd chelator lipid with avidin and biotinylated antibody.See, for example, Sipkins et al., Nature Med., 4:623-626 (1998).

Suitable fluorescent proteins include, but are not limited to, greenfluorescent protein (GFP) or variants thereof, blue fluorescent variantof GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescentvariant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhancedYFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine,GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP),destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet,mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2,t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP,Kaede protein and kindling protein, Phycobiliproteins andPhycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrinand Allophycocyanin. Other examples of fluorescent proteins includemHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry,mCherry, mGrapel, mRaspberry, mGrape2, and/or mPlum (Shaner et al.(2005) Nat. Methods 2:905-909). Any of a variety of fluorescent andcolored proteins from Anthozoan species, as described in, e.g., Matz etal. (1999) Nature Biotechnol. 17:969-973, is suitable for use.

In some embodiments, a humanized anti-factor Bb antibody is conjugatedto a therapeutic agent. Any of the humanized anti-factor Bb antibodiesdisclosed herein can be used to form an antibody-agent conjugate. Theagent can be attached to the N terminus of the light chain, the Cterminus of the light chain, the N terminus of the heavy chain, or the Cterminus of the heavy chain. In some embodiments, the agent is attachedto the hinge of the antibody or to at least one other sites on theantibody. For a single chain antibody, the agent can be attached to theN or C terminus of the single chain antibody. The agent can beconjugated to the antibody directly or via a linker using techniquesknown to those skilled in the art. The linker can be cleavable ornon-cleavable. Examples of such therapeutic agents (e.g., for use intherapy) are known to those skilled in the art.

In some embodiments, a humanized anti-factor Bb antibody is linked to(e.g., covalently or non-covalently linked) a fusion partner, e.g., aligand; an epitope tag; a peptide; and/or a protein other than anantibody. Suitable fusion partners include peptides and polypeptidesthat confer enhanced stability in vivo (e.g., enhanced serum half-life);provide ease of purification, e.g., (His)n, e.g., 6His; provide forsecretion of the fusion protein from a cell; provide an epitope tag,e.g., GST, hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO: 55), FLAG(e.g., DYKDDDDK; SEQ ID NO: 56), and/or c-myc (e.g., EQKLISEEDL; SEQ IDNO: 57); provide a detectable signal, e.g., an enzyme that generates adetectable product (e.g., β-galactosidase, luciferase), or a proteinthat is itself detectable, e.g., a green fluorescent protein, a redfluorescent protein, a yellow fluorescent protein, etc.; and/or providesfor multimerization, e.g., a multimerization domain such as an Fcportion of an immunoglobulin. The fusion can also include an affinitydomain, including peptide sequences that can interact with a bindingpartner, e.g., such as one immobilized on a solid support, useful foridentification or purification. Consecutive single amino acids, such ashistidine, when fused to a protein, can be used for one-steppurification of the fusion protein by high affinity binding to a resincolumn, such as nickel sepharose. Exemplary affinity domains includeHis5 (HHHHH) (SEQ ID NO: 58), His×6 (HHHHHH) (SEQ ID NO:59), C-myc(EQKLISEEDL) (SEQ ID NO:60), Flag (DYKDDDDK) (SEQ ID NO:61), StrepTag(WSHPQFEK) (SEQ ID NO:62), hemagglutinin, e.g., HA Tag (YPYDVPDYA; SEQID NO:63), glutathinone-S-transferase (GST), thioredoxin, cellulosebinding domain, RYIRS (SEQ ID NO:66), Phe-His-His-Thr (SEQ ID NO:64),chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-end RNA tag,WEAAAREACCRECCARA (SEQ ID NO:65), metal binding domains, e.g., zincbinding domains or calcium binding domains such as those fromcalcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B,myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin,hippocalcin, frequenin, caltractin, calpain large-subunit, S100proteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin,inteins, biotin, streptavidin, MyoD, leucine zipper sequences, andmaltose binding protein.

Methods of Producing Humanized Anti-Factor Bb Antibodies

In some embodiments, fully human antibodies can be obtained by usingcommercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are XenomouseR™fromAmgen, Inc. (Fremont, Calif.) and HuMAb-MouseR™ and TC Mouse™ fromMedarex, Inc. (Princeton, N.J.) or H2L2 mice from Harbour

Antibodies BV (Holland). In another alternative, antibodies may be maderecombinantly by phage display or yeast technology. See, for example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winteret al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, the phagedisplay technology (McCafferty et al., (1990) Nature 348:552-553) can beused to produce human antibodies and antibody fragments in vitro, fromimmunoglobulin variable (V) domain gene repertoires from unimmunizeddonors.

Antigen-binding fragments of an intact antibody (full-length antibody)can be prepared via routine methods. For example, F(ab′)₂ fragments canbe produced by pepsin digestion of an antibody molecule, and Fabfragments that can be generated by reducing the disulfide bridges ofF(ab′)₂ fragments. Genetically engineered antibodies, such as humanizedantibodies, chimeric antibodies, single-chain antibodies, and bispecificantibodies, can be produced via, e.g., conventional recombinanttechnology. In one example, DNA encoding a monoclonal antibodiesspecific to a target antigen can be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the monoclonal antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoat least one expression vector, which is then transfected into hostcells such as Escherichia coli (E. coli) cells, simian COS cells,Chinese hamster ovary (CHO) cells, human HEK293 cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in recombinant host cells. See, e.g.,PCT Publication No. WO 87/04462. The DNA can then be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences,Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851, or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. In that manner,genetically engineered antibodies, such as “chimeric” or “hybrid”antibodies; can be prepared that have the binding specificity of atarget antigen.

A single-chain antibody can be prepared via recombinant technology, forexample, by linking a nucleotide sequence coding for a heavy chainvariable region and a nucleotide sequence coding for a light chainvariable region. In some embodiments, a flexible linker is incorporatedbetween the two variable regions.

Alternatively, techniques described for the production of single chainantibodies (U. S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted, forexample, to produce a phage or yeast scFv library and scFv clonesspecific to factor Bb can be identified from the library followingroutine procedures. Positive clones can be subjected to furtherscreening to identify those that has high factor Bb binding affinity.

In some embodiments, a humanized anti-factor Bb antibody is prepared byrecombinant technology as exemplified below. Nucleic acids encoding theheavy and light chain of an anti-factor Bb antibody as described hereincan be cloned into one expression vector, each nucleotide sequence beingin operable linkage to a suitable promoter. In one example, each of thenucleotide sequences encoding the heavy chain and light chain is inoperable linkage to a distinct promoter. Alternatively, the nucleotidesequences encoding the heavy chain and the light chain can be inoperable linkage with a single promoter, such that both heavy and lightchains are expressed from the same promoter. When necessary, an internalribosomal entry site (IRES) can be inserted between the heavy chain andlight chain encoding sequences.

In some examples, the nucleotide sequences encoding the two chains ofthe antibody are cloned into two vectors, which can be introduced intothe same or different cells. When the two chains are expressed indifferent cells, each of them can be isolated from the host cellsexpressing such and the isolated heavy chains and light chains can bemixed and incubated under suitable conditions allowing for the formationof the antibody.

Generally, a nucleic acid sequence encoding one or all chains of anantibody can be cloned into a suitable expression vector in operablelinkage with a suitable promoter using methods known in the art. Forexample, the nucleotide sequence and vector can be contacted, undersuitable conditions, with a restriction enzyme to create complementaryends on each molecule that can pair with each other and be joinedtogether with a ligase. Alternatively, synthetic nucleic acid linkerscan be ligated to the termini of a gene. These synthetic linkers containnucleic acid sequences that correspond to a particular restriction sitein the vector. The selection of expression vectors/promoters woulddepend on the type of host cells for use in producing the antibodies.

A variety of promoters can be used for expression of the antibodiesdescribed herein, including, but not limited to, cytomegalovirus (CMV)intermediate early promoter, a viral LTR such as the Rous sarcoma virusLTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E.coli lac UV promoter, and the herpes simplex tk virus promoter.

Regulatable promoters can also be used. Such regulatable promotersinclude those using the lac repressor from E. coli as a transcriptionmodulator to regulate transcription from lac operator bearing mammaliancell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those usingthe tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc.Natl. Acad. Sci. USA 89:5547-555115 (1992); Yao, F. et al., Human GeneTherapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad.Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP16or p65 using astradiol, RU486, diphenol murislerone, or rapamycin.Inducible systems are available from Invitrogen, Clontech and Ariad,among others.

Regulatable promoters that include a repressor with the operon can beused. In one embodiment, the lac repressor from E. coli can function asa transcriptional modulator to regulate transcription from lacoperator-bearing mammalian cell promoters [M. Brown et al., Cell,49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl.Acad. Sci. USA, 89:5547-5551(1992)] combined the tetracycline repressor(tetR) with the transcription activator (VP 16) to create atetR-mammalian cell transcription activator fusion protein, tTa(tetR-VP16), with the tetO bearing minimal promoter derived from thehuman cytomegalovirus (hCMV) promoter to create a tetR-tet operatorsystem to control gene expression in mammalian cells. In one embodiment,a tetracycline inducible switch is used. The tetracycline repressor(tetR) alone, rather than the tetR-mammalian cell transcription factorfusion derivatives can function as potent trans-modulator to regulategene expression in mammalian cells when the tetracycline operator isproperly positioned downstream for the TATA element of the CMVIEpromoter (Yao et al., Human Gene Therapy). One particular advantage ofthis tetracycline inducible switch is that it does not require the useof a tetracycline repressor-mammalian cells transactivator or repressorfusion protein, which in some instances can be toxic to cells (Gossen 5et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al.,Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)), to achieve itsregulatable effects.

Additionally, the vector can contain, for example, some or all of thefollowing: a selectable marker gene, such as the neomycin gene forselection of stable or transient transfectants in mammalian cells;enhancer/promoter sequences from the immediate early gene of human CMVfor high levels of transcription; transcription termination and RNAprocessing signals from SV40 for mRNA stability; SV40 polyoma origins ofreplication and ColE1 for proper episomal replication; internal ribosomebinding sites (IRESes), versatile multiple cloning sites; and T7 and SP6RNA promoters for in vitro transcription of sense and antisense RNA.Suitable vectors and methods for producing vectors containing transgenesare well known and available in the art.

Examples of polyadenylation signals useful to practice the methodsdescribed herein include, but are not limited to, human collagen Ipolyadenylation signal, human collagen II polyadenylation signal, andSV40 polyadenylation signal.

At least one vector (e.g., expression vectors) comprising nucleic acidsencoding any of the antibodies may be introduced into suitable hostcells for producing the antibodies. The host cells can be cultured undersuitable conditions for expression of the antibody or any polypeptidechain thereof. Such antibodies or polypeptide chains thereof can berecovered by the cultured cells (e.g., from the cells or the culturesupernatant) via a conventional method, e.g., affinity purification. Ifnecessary, polypeptide chains of the antibody can be incubated undersuitable conditions for a suitable period of time allowing forproduction of the antibody.

In some embodiments, methods for preparing an antibody described hereininvolve a recombinant expression vector that encodes both the heavychain and the light chain of an anti-factor Bb antibody, as alsodescribed herein. The recombinant expression vector can be introducedinto a suitable host cell (e.g., a dihydrofolate reductase (DHFR)-CHOcell) by a conventional method, e.g., calcium phosphate mediatedtransfection. Positive transformant host cells can be selected andcultured under suitable conditions allowing for the expression of thetwo polypeptide chains that form the antibody, which can be recoveredfrom the cells or from the culture medium. When necessary, the twochains recovered from the host cells can be incubated under suitableconditions allowing for the formation of the antibody.

In some embodiments, two recombinant expression vectors are provided,one encoding the heavy chain of the anti-factor Bb antibody and theother encoding the light chain of the anti-factor Bb antibody. Both ofthe two recombinant expression vectors can be introduced into a suitablehost cell (e.g., DHFR-CHO cell) by a conventional method, e.g., calciumphosphate-mediated transfection.

Alternatively, each of the expression vectors can be introduced into asuitable host cells.

Positive transformants can be selected and cultured under suitableconditions allowing for the expression of the polypeptide chains of theantibody. When the two expression vectors are introduced into the samehost cells, the antibody produced therein can be recovered from the hostcells or from the culture medium. If necessary, the polypeptide chainscan be recovered from the host cells or from the culture medium and thenincubated under suitable conditions allowing for formation of theantibody. When the two expression vectors are introduced into differenthost cells, each of them can be recovered from the corresponding hostcells or from the corresponding culture media. The two polypeptidechains can then be incubated under suitable conditions for formation ofthe antibody.

Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recovery of the antibodiesfrom the culture medium. For example, some antibodies can be isolated byaffinity chromatography with a Protein A or Protein G coupled matrix.

Any of the nucleic acids encoding the heavy chain, the light chain, orboth of an anti-factor Bb antibody as described herein (e.g., asprovided in Tables 2 and 3), vectors (e.g., expression vectors)containing such; and host cells comprising the vectors are within thescope of the present disclosure.

Pharmaceutical Compositions and Therapeutic Methods

Other aspects of the present disclosure provide compositions, includingpharmaceutical compositions comprising any one of the humanizedanti-factor Bb antibodies described herein. In general, a pharmaceuticalcomposition, also referred to herein as a formulation, comprises aneffective amount of any one of the humanized anti-factor Bb antibodiesdescribed herein. An “effective amount” means a dosage sufficient toproduce a desired result, e.g., reduction in an adverse symptomassociated with a complement-mediated disease or disorder, ameliorationof a symptom of a complement-mediated disease or disorder, slowingprogression of a complement-mediated disease or disorder, etc.Generally, the desired result is at least a reduction in a symptom of acomplement-mediated disease or disorder, as compared to a control.

In a method of the present disclosure, a humanized anti-factor Bbantibody can be administered to a subject using a convenient meanscapable of resulting in the desired therapeutic effect or diagnosticeffect. Thus, a humanized anti-factor Bb antibody can be incorporatedinto a variety of formulations for therapeutic administration. Moreparticularly, a humanized anti-factor Bb antibody can be formulated intopharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers, pharmaceutically acceptablediluents, or other pharmaceutically acceptable excipients. In someembodiments, a pharmaceutical composition comprises a humanizedanti-factor Bb antibody and a pharmaceutically acceptable excipient.

In pharmaceutical dosage forms, a humanized anti-factor Bb antibody canbe administered in the form of their pharmaceutically acceptable salts,or they can also be used alone or in appropriate association, as well asin combination, with other pharmaceutically active compounds. Thefollowing methods and excipients are merely exemplary and are in no waylimiting.

For oral preparations, a humanized anti-factor Bb antibody can be usedalone or in combination with appropriate additives to make tablets,powders, granules or capsules.

A humanized anti-factor Bb antibody can be formulated into preparationsfor injection by dissolving, suspending or emulsifying the antibody inan aqueous or nonaqueous solvent; and if desired, with conventionaladditives such as solubilizers, isotonic agents, suspending agents,emulsifying agents, stabilizers and preservatives.

Pharmaceutical compositions comprising a humanized anti-factor Bbantibody are prepared by mixing a humanized anti-factor Bb antibodyhaving the desired degree of purity with optional physiologicallyacceptable carriers, other excipients, stabilizers, surfactants, buffersand/or tonicity agents. Acceptable carriers, other excipients and/orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed. In some embodiments, the compositions comprise a buffer, anantioxidant, an amino acid or a combination thereof. The pharmaceuticalcomposition can be in a liquid form, a lyophilized form or a liquid formreconstituted from a lyophilized form, wherein the lyophilizedpreparation is to be reconstituted with a sterile solution prior toadministration. The standard procedure for reconstituting a lyophilizedcomposition is to add back a volume of pure water (typically equivalentto the volume removed during lyophilization); however, solutionscomprising antibacterial agents can be used for the production ofpharmaceutical compositions for parenteral administration; see also Chen(1992) Drug Dev Ind Pharm 18, 1311-54.

A tonicity agent can be included in the antibody formulation to modulatethe tonicity of the formulation. In some embodiments, the aqueousformulation is isotonic, although hypertonic or hypotonic solutions canbe suitable. The term “isotonic” denotes a solution having the sametonicity as some other solution with which it is compared, such as aphysiological salt solution or serum.

A surfactant can also be added to the antibody formulation to reduceaggregation of the formulated antibody and/or minimize the formation ofparticulates in the formulation and/or reduce adsorption. Alyoprotectant can also be added in order to protect the labile activeingredient (e.g., a protein) against destabilizing conditions during thelyophilization process.

In some embodiments, a subject formulation includes a humanizedanti-factor Bb antibody, and at least one of the above-identified agents(e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and isessentially free of at least one preservative.

A humanized anti-factor Bb antibody can be utilized in aerosolformulation to be administered via inhalation. A humanized anti-factorBb antibody can be formulated into pressurized acceptable propellants.

Furthermore, a humanized anti-factor Bb antibody can be made intosuppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases. A humanized anti-factor Bb antibody can beadministered rectally via a suppository.

Other modes of administration will also find use with a method of thepresent disclosure. For instance, a humanized anti-factor Bb antibodycan be formulated in suppositories and, in some embodiments, aerosol andintranasal compositions. For suppositories, the vehicle composition mayinclude traditional binders and carriers.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed. The nasal formulations can also contain apreservative. A surfactant can be present to enhance absorption of ahumanized anti-factor Bb antibody by the nasal mucosa.

A humanized anti-factor Bb antibody can be administered as an injectableformulation. Typically, injectable compositions are prepared as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation can also be emulsified, or the antibody encapsulated inliposome vehicles.

In some embodiments, a humanized anti-factor Bb antibody is formulatedin a controlled release formulation. Controlled release within the scopeof the present disclosure can be taken to mean any one of a number ofextended-release dosage forms.

A humanized anti-factor Bb antibody is administered to a subject using amethod and route suitable for drug delivery, including in vivo and exvivo methods, as well as systemic and localized routes ofadministration.

Conventional and pharmaceutically acceptable routes of administrationinclude, but are not limited to, intranasal, intramuscular,intratracheal, intrathecal, intracranial, subcutaneous, intradermal,topical, intravenous, intraperitoneal, intraarterial (e.g., via thecarotid artery), spinal or brain delivery, rectal, nasal, oral, andother enteral and parenteral routes of administration.

An antibody of the present disclosure can be administered to a subjectusing any available conventional methods and routes suitable fordelivery of conventional drugs, including systemic or localized routes.In general, routes of administration contemplated by the presentdisclosure include, but are not necessarily limited to, enteral,parenteral, or inhalational routes.

In some embodiments, a humanized anti-factor Bb antibody is administeredby injection and/or delivery, e.g., to a site in a brain artery ordirectly into brain tissue. A humanized anti-factor Bb antibody can alsobe administered directly to a target site e.g., by biolistic delivery tothe target site.

A variety of subjects may be treated in accordance with the methodsprovided herein. Generally, such subjects are “mammals” or “mammalian,”where these terms are used broadly to describe organisms which arewithin the class Mammalia, including the orders carnivores (e.g., cats),herbivores (e.g., cattle, horses, and sheep), omnivores (e.g., dogs,goats, and pigs), rodentia (e.g., mice, guinea pigs, and rats), andprimates (e.g., humans, chimpanzees, and monkeys). In some embodiments,a subject has a complement system, such as a mammal, fish, orinvertebrate. In some embodiments, a subject is a complementsystem-containing mammal, fish, or invertebrate companion animal,agricultural animal, work animal, zoo animal, or lab animal. In someembodiments, a subject is a human.

“Treatment” refers to at least an amelioration of the symptomsassociated with pathological condition afflicting a subject, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g., symptom, associated with thepathological condition being treated, such as a complement-mediateddisease or disorder. As such, treatment also includes situations wherethe pathological condition, or at least symptoms and/or secondaryeffects associated therewith, are completely inhibited, e.g., preventedfrom happening, or stopped, e.g., terminated, such that subject nolonger suffers from the pathological condition, or at least the symptomsthat characterize the pathological condition.

In some embodiments, a “subject” is a mammal, including, but not limitedto, murines (rats, mice), non-human primates, humans, canines, felines,ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.Also encompassed by these terms are any animal that has a complementsystem, such as mammals, fish, and some invertebrates. As such theseterms include complement system-containing mammal, fish, andinvertebrate companion animals, agricultural animals, work animals, zooanimals, and lab animals.

A “biological sample” encompasses a variety of sample types obtainedfrom a subject and can be used in a diagnostic or monitoring assay. Thedefinition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such aspolynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples. The term“biological sample” includes urine, saliva, cerebrospinal fluid,interstitial fluid, ocular fluid, synovial fluid, and/or blood fractionssuch as plasma and serum. The term “biological sample” also includessolid tissue samples, tissue culture samples, and cellular samples.

The present disclosure provides a device or container suitable forcontaining a composition comprising a humanized anti-factor Bb antibodyfor administration to a subject. For example, a humanized anti-factor Bbantibody can be disposed within a container suitable for containing apharmaceutical composition. The container can be, for example, a bottle(e.g., with a closure device, such as a cap), a blister pack (e.g.,which can provide for enclosure of at least one doses per blister), avial, flexible packaging (e.g., sealed Mylar or plastic bags), an ampule(for single doses in solution), a dropper, a syringe, thin film, and/ora tube. In some embodiments, a container, such as a sterile container,comprises a subject pharmaceutical composition. In some embodiments, thecontainer is a bottle or a syringe. In some embodiments, the containeris a bottle. In some embodiments, the container is a syringe. In someembodiments, the device is an injectable device, such as a syringe(e.g., a pre-filled syringe), a pen (e.g., a pre-filled pen), or anelectronic injection device (e-Devices).

The present disclosure provides methods of treating acomplement-mediated disease or disorder. The methods generally involveadministering an effective amount of a humanized anti-factor Bb antibodyof the present disclosure to a subject in need thereof. In someembodiments, administration of a humanized anti-factor Bb antibodymodulates the activity of the complement pathway activity in a cell, atissue, or a fluid of a subject, and treats the complement-mediateddisease or disorder.

An “effective amount” refers to the amount of an anti-complement factorBb antibody that, when administered to a mammal or other subject fortreating a disease, is sufficient to effect such treatment for thedisease. The “therapeutically effective amount” will vary depending onthe anti-complement Bb antibody, the disease and its severity and theage, weight, etc., of the subject to be treated.

In some embodiments, an effective amount of a humanized anti-factor Bbantibody of the present disclosure is an amount that is effective toreduce or inhibit complement pathway activity in a cell, tissue, orfluid of the subject.

In some embodiments, an effective amount of a humanized anti-factor Bbantibody of the present disclosure is an amount that is effective toreduce or inhibit formation of MAC in a cell, tissue, or fluid of thesubject.

In some embodiments, an effective amount of a humanized anti-factor Bbantibody of the present disclosure is an amount that is effective toreduce or inhibit C3b/Bb-mediated cleavage of C3 in a cell, tissue, orfluid of the subject.

In some embodiments, an effective amount of a humanized anti-factor Bbantibody of the present disclosure is an amount that is effective toreduce or inhibit C3b/Bb-mediated cleavage of C3, and thereby to reduceproduction of a C3 cleavage product.

In some embodiments, an effective amount of a humanized anti-factor Bbantibody of the present disclosure is an amount that is effective toreduce or inhibit complement AP-mediated lysis of a cell in the subject.

In some embodiments, an effective amount of the humanized anti-factor Bbantibody of the present disclosure is an amount that is effective toreduce or inhibit complement AP-mediated hemolysis in a cell, tissue, orfluid (e.g., RBC-containing fluid) of the subject.

In some embodiments, an effective amount of a humanized anti-factor Bbantibody of the present disclosure is an amount that is effective toreduce or inhibit production of an anaphylatoxin.

In some embodiments, an effective amount of a humanized anti-factor Bbantibody of the present disclosure is an amount that is effective toreduce or inhibit AP-mediated deposition of C3b, C3d, or other C3 splitproduct on a cell or tissue in the subject. In some embodiments, aneffective amount of a humanized anti-factor Bb antibody of the presentdisclosure is an amount that is effective to reduce or inhibitAP-mediated C3b deposition on a cell or tissue in the subject.

In some embodiments, an effective amount of a humanized anti-factor Bbantibody of the present disclosure is an amount that is effective toreduce or inhibit AP-mediated deposition of C3b, C3d, or other C3 splitproduct on RBCs in the subject.

In some embodiments, an effective amount of a humanized anti-factor Bbantibody of the present disclosure is an amount that is effective toreduce or inhibit AP-mediated C3b deposition on RBCs in the subject.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure, when administered in at least one doses to a subject in needthereof, reduces the amount of factor Bb in circulation in the subject.For example, a humanized anti-factor Bb antibody of the presentdisclosure, when administered in at least one doses to a subject in needthereof, may reduce the amount of factor Bb in circulation in thesubject by at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95%, compared to theamount of factor Bb in circulation in the subject in the absence ofadministering the humanized anti-factor Bb antibody, or compared to theamount of factor Bb in circulation in the subject before administrationof the humanized anti-factor Bb antibody.

In some embodiments, a humanized anti-factor Bb antibody of the presentdisclosure, when administered in at least one doses to a subject in needthereof, reduces the amount of factor Bb in plasma in the subject. Forexample, a humanized anti-factor Bb antibody of the present disclosure,when administered in at least one doses to a subject in need thereof,may reduce the amount of factor Bb in plasma in the subject by at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95%, compared to the amount of factor Bbin plasma in the subject in the absence of administering the humanizedanti-factor Bb antibody, or compared to the amount of factor Bb inplasma in the subject before administration of the humanized anti-factorBb antibody.

In some embodiments, a method of the present disclosure to treat asubject having a complement-mediated disease or disorder comprisesadministering to the subject a humanized anti-factor Bb antibody of thepresent disclosure or a pharmaceutical composition comprising: a) ahumanized anti-factor Bb antibody of the present disclosure; and b) apharmaceutically acceptable excipient suitable for administration tosuch subject. In some embodiments, the subject is a mammal. In someembodiments, the subject is a human. Administering can be by any routeknown to those skilled in the art, including those disclosed herein. Insome embodiments, administering is intravenous. In some embodiments,administering is intrathecal. In some embodiments, administering isintramuscular. In some embodiments, administering is subcutaneous.

Complement-mediated diseases and disorders that are suitable fortreatment with a humanized anti-factor Bb antibody of the presentdisclosure include diseases and disorders associated with thealternative complement pathway. Studies in preclinical animal models andclinical trials indicate alternative pathway plays an important role inthe development of tissue injury and pathogenesis of several conditions(Holers et al., Immunological Reviews 223: 300-316; Cao et al., (2016)Haematologica 101(11): 1319-1326; Schubart et al., (2019) PNAS 116(16):7926-7931; Thurman, (2015) Am J Kidney Dis 65(1): 156-168; Vriese etal., (2015) Am J Kidney Dis 65(1): 156-168; and Gold et al., (2006) Nat.Genet. 38(4): 458-462). In some embodiments, complement-mediateddiseases that are suitable for treatment with a humanized anti-factor Bbantibody of the present disclosure include, but are not limited to, IgAnephropathy (Berger's disease), atypical hemolytic uremic syndrome(aHUS), paroxysmal nocturnal hemoglobinuria (PNH), idiopathicthrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura(TTP), lupus nephritis, ANCA vasculitis, membranous nephropathy, C3glomerulonephritis (C3GN), focal segmental glomerulosclerosis (FSGS),multiple sclerosis, macular degeneration, age-related maculardegeneration (AMD), rheumatoid arthritis, antiphospholipid antibodysyndrome, asthma, ischemia-reperfusion injury, Type IImembranoproliferative GN, spontaneous fetal loss, Pauci-immunevasculitis, epidermolysis bullosa, recurrent fetal loss, and traumaticbrain injury.

A humanized anti-factor Bb antibody of the present disclosure can beadministered to a subject in need thereof alone (e.g., as monotherapy);or in combination therapy with at least one additional therapeuticagents.

“In combination with” as used herein refers to uses where, for example,the first compound is administered during the entire course ofadministration of the second compound; where the first compound isadministered for a period of time that is overlapping with theadministration of the second compound, e.g. where administration of thefirst compound begins before the administration of the second compoundand the administration of the first compound ends before theadministration of the second compound ends; where the administration ofthe second compound begins before the administration of the firstcompound and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe first compound begins before administration of the second compoundbegins and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe second compound begins before administration of the first compoundbegins and the administration of the first compound ends before theadministration of the second compound ends. As such, “in combination”can also refer to regimen involving administration of two or morecompounds.” In combination with” as used herein also refers toadministration of two or more compounds that can be administered in thesame or different formulations, by the same of different routes, and inthe same or different dosage form type.

Subjects suitable for treatment with a humanized anti-factor Bb antibodyinclude subjects who have been diagnosed as having a complement-mediateddisease or disorder; subjects at greater risk than the generalpopulation for developing a complement-mediated disease or disorder(e.g., subjects having a genetic predisposition to developing acomplement-mediated disease or disorder). Also included are subjectshaving any one of the complement-mediated diseases or disorders listedhereinabove. In some embodiments, the subject is an adult human. In someembodiments, the subject is a human child.

EXAMPLES Example 1 Humanization of a Mouse Monoclonal Anti-Factor BbAntibody

Variable region genes from the parent anti-factor Bb antibody (seeTable 1) hybridoma were amplified, cloned and sequenced, resulting inthe identification of a single unique VH domain and a single unique Vκdomain.

Initially, three humanized VH regions and five humanized Vκ regionsdesigned using COMPOSITE HUMAN ANTIBODY™ technology were cloned intoIgG4v1 heavy chain and kappa light chain vectors. The parent antibody,two control antibodies and all 15 humanized antibody combinations wereexpressed transiently in HEK EBNA cells.

In order to assess the binding of all humanized variants, single cyclekinetic analysis was performed on supernatants from transfected cellcultures. Kinetic experiments were performed on a Biacore T200 (serialno. 1909913) running Biacore T200 Control software V2.0.1 and Evaluationsoftware V3.0 (GE Healthcare, Uppsala, Sweden). All single cycle kineticexperiments were run at 25° C. with HBS-P+running buffer (pH 7. 4) (GEHealthcare, Little Chalfont, UK).

Antibodies were diluted in running buffer to a final concentration of 1μg/ml, based on concentrations assessed by ELISA titer. At the start ofeach cycle, antibodies were loaded onto Fc2, Fc3 and Fc4 of the ProteinA chip (GE Healthcare, Little Chalfont, UK). IgGs were captured at aflow rate of 10 μl/min to give an immobilization level (RL) of ˜63 RU,the theoretical value to obtain an Rmax of ˜50 RU. The surface was thenallowed to stabilize. Single cycle kinetic data was obtained with factorBb (CompTech, Tyler, USA) as the analyte at a flow rate of 30 μl/min tominimize any potential mass transport limitations. Multiple repeats withthe reference chimeric antibody were performed to check the stability ofthe surface and analyte over the kinetic cycles. The signal from thereference channel Fc1 (no antibody) was subtracted from that of Fc2, Fc3and Fc4 to correct for differences in non-specific binding to areference surface. A four point, two-fold dilution range from 0. 78 nMto 6. 25 nM factor Bb without regeneration between each concentrationwas used. The association phase for the four injections of increasingconcentrations of factor Bb was monitored for 200 seconds each time anda single dissociation phase was measured for 200 seconds following thelast injection of factor Bb. Regeneration of the Protein A surface wasconducted using two injections of 10 mM glycine-HCL pH 1. 5.

The sensorgrams and fitted data for the single cycle kinetics are shownin FIGS. 1A-1D and the kinetic parameters measured for the interactionof factor Bb with each antibody are shown in Table 7. The relative K_(D)was calculated by dividing the K_(D) of the VH0/Vκ0 reference antibodyby that of the humanized variant assayed in the same experiment.

TABLE 7 Single Cycle Kinetic Parameters of the Humanized Variants andReference Antibodies Binding to Factor Bb. ka Chi² Relative Antibody(1/Ms) kd (1/s) KD (M) (RU²) KD VH0/Vκ0 5.85 × 10⁵ 1.03 × 10⁻³ 1.76 ×10⁻⁹ 0.0134  1.00 (Chimeric) VH0/Vκ1 5.86 × 10⁵ 1.57 × 10⁻³ 2.68 × 10⁻⁹0.00688 1.52 (control) VH1/Vκ0 3.76 × 10⁵ 1.16 × 10⁻³ 3.09 × 10⁻⁹0.00605 1.76 (control) VH1/Vκ1 5.74 × 10⁵ 2.87 × 10⁻³ 5.01 × 10⁻⁹0.00228 2.85 VH1/Vκ2 4.81 × 10⁵ 2.51 × 10⁻³ 5.22 × 10⁻⁹ 0.00381 2.97VH1/Vκ3 6.75 × 10⁵ 2.63 × 10⁻³ 3.90 × 10⁻⁹ 0.00488 2.22 VH1/Vκ4 4.51 ×10⁵ 2.94 × 10⁻³ 6.52 × 10⁻⁹ 0.0029  3.70 VH1/Vκ5 2.25 × 10⁵ 2.01 × 10⁻³8.96 × 10⁻⁹ 0.00803 5.09 VH2/Vκ1 1.80 × 10⁵ 3.73 × 10⁻³ 2.07 × 10⁻⁸0.00991 11.78  VH2/Vκ2 2.34 × 10⁵ 3.26 × 10⁻³ 1.39 × 10⁻⁸ 0.00327 7.90VH2/Vκ3 2.52 × 10⁵ 3.07 × 10⁻³ 1.22 × 10⁻⁸ 0.00259 6.93 VH2/Vκ4 3.39 ×10⁵ 3.68 × 10⁻³ 1.09 × 10⁻⁸ 0.00377 6.19 VH2/Vκ5 2.25 × 10⁵ 3.75 × 10⁻³1.66 × 10⁻⁸ 0.00241 9.43 VH3/Vκ1 2.91 × 10⁵ 4.04 × 10⁻³ 1.39 × 10⁻⁸0.00393 7.90 VH3/Vκ2 2.36 × 10⁵ 3.77 × 10⁻³ 1.60 × 10⁻⁸ 0.0019  9.09VH3/Vκ3 2.20 × 10⁵ 3.29 × 10⁻³ 1.49 × 10⁻⁸ 0.00359 8.47 VH3/Vκ4 2.67 ×10⁵ 4.60 × 10⁻³ 1.73 × 10⁻⁸ 0.00332 9.83 VH3/Vκ5 2.85 × 10⁵ 4.51 × 10⁻³1.58 × 10⁻⁸ 0.00257 8.98

Biacore analysis showed that all humanized variants bound to factor Bb,however, in all cases the relative K_(D) was greater than two-folddifferent than the chimeric suggesting that some binding affinity hadbeen lost. In order to address this, an additional four heavy chain (VH4to VH7) and two light chain (Vκ6 to Vκ7) sequences were designed andcloned into the appropriate expression vector. Variant sequences areshown in Tables 2 and 3.

Expression and Single Cycle Kinetic Analysis of Redesigned Variants

Five control antibodies (VH0/Vκ6, VH0/Vκ7, VH5/Vκ0, VH6/Vκ0, VH7/Vκ0)and combinations of humanized heavy and light chains (a total of eighthumanized pairings, Table 8) were transiently transfected into HEK EBNAadherent cells (ATCC® Cat. No. CRL-10852™) using a PEI transfectionmethod. IgG supernatant titres were monitored by IgG ELISA (Table 9) andtransfections were cultured for up to 10 days prior to harvestingsupernatants.

TABLE 8 Antibody Titers of Variants Produced by Transient Transfection(μg/mL). Vκ0 Vκ6 Vκ7 VH0 — 60.6 29.9 VH4 — 4.71 4.75 VH5 26.9 38.1 35.5VH6 5.8 8.5 9.7 VH7 17.9 36.1 39.8

Single cycle kinetics using cell culture supernatants were performed asdescribed above. The sensorgrams and fitted data for the single cyclekinetics are shown in FIGS. 2A-2C. All variants were shown to bind tofactor Bb. Single cycle kinetics data (Table 9) demonstrated that fiveantibodies (VH4/Vκ6, VH4/Vκ7, VH6/Vκ6 VH6/Vκ7 and VH7/Vκ7) bound tofactor Bb within ˜two-fold of the reference chimeric antibody. Therelative K_(D) was calculated by dividing the K_(D) of the VH0/Vκ0reference antibody by that of the humanized variant assayed in the sameexperiment.

TABLE 9 Single Cycle Kinetic Parameters of the Humanized Variants andReference Antibodies Binding to Factor Bb. Chi² Relative Antibody ka(1/Ms) kd (1/s) KD (M) (RU²) KD VH0/Vκ0 5.35 × 10⁵ 9.49 × 10⁻⁴ 1.77 ×10⁻⁹ 0.00854  1.00 VH0/Vκ3 5.63 × 10⁵ 1.28 × 10⁻³ 2.27 × 10⁻⁹ 0.00587 1.28 VH0/Vκ6 5.34 × 10⁵ 1.26 × 10⁻³ 2.37 × 10⁻⁹ 0.00766  1.34 VH0/Vκ74.96 × 10⁵ 1.05 × 10⁻³ 2.11 × 10⁻⁹ 0.00335  1.19 VH4/Vκ6 3.87 × 10⁵ 8.59× 10⁻⁴ 2.22 × 10⁻⁹ 0.00473  1.25 VH4/Vκ7 3.86 × 10⁵ 9.03 × 10⁻⁴ 2.34 ×10⁻⁹ 0.00497  1.32 VH5/Vκ0 3.49 × 10³ 1.45 × 10⁻³ 4.16 × 10⁻⁷ 0.11  235.03 VH5/Vκ6 1.33 × 10⁵ 1.73 × 10⁻² 1.30 × 10⁻⁷ 0.173   73.45 VH5/Vκ73.20 × 10³ 1.66 × 10⁻³ 5.21 × 10⁻⁷ 0.153  294.35 VH6/Vκ0 5.98 × 10⁵ 1.97× 10⁻³ 3.29 × 10⁻⁹ 0.00485  1.86 VH6/Vκ6 4.12 × 10⁵ 1.58 × 10⁻³ 3.84 ×10⁻⁹ 0.00588  2.17 VH6/Vκ7 6.30 × 10⁵ 2.21 × 10⁻³ 3.51 × 10⁻⁹ 0.00377 1.98 VH7/Vκ0 2.74 × 10⁵ 2.07 × 10⁻³ 7.53 × 10⁻⁹ 0.00634  4.25 VH7/Vκ61.68 × 10⁵ 3.03 × 10⁻³ 1.80 × 10⁻⁸ 0.00587  10.17 VH7/Vκ7 4.83 × 10⁶1.17 × 10⁻² 2.42 × 10⁻⁹ 0.0384   1.37

Purification of Antibodies

VH4/Vκ6, VH4/Vκ7, VH6/Vκ6 VH6/Vκ7 and VH7/Vκ7 as well as the chimericantibody were purified from cell culture supernatants on Protein Asepharose columns (GE Healthcare, Little Chalfont, UK), followed by sizeexclusion chromatography using a 16/60 Superdex 200 column (GEHealthcare, Little Chalfont, UK) using PBS pH 7. 4 as the mobile phase.Antibodies were quantified by OD_(280nm) using an extinction coefficientbased on the predicted amino acid sequence. Reduced antibodies wereanalyzed using SDS-PAGE by loading 1 μg of each antibody on the gel(FIG. 3) and bands corresponding to the profile of a typical antibodywere observed.

Multicycle Kinetics Analysis of Antibodies

In order to establish an accurate affinity for factor Bb, multicyclekinetics analysis was performed on the purified chimeric antibody andthe five lead antibodies using a Biacore T200 (serial no. 1909913)instrument running Biacore T200 Evaluation Software V3.0.1 (Uppsala,Sweden). Antibodies were diluted in running buffer to a finalconcentration of 0.5 μg/ml. At the start of each cycle, antibodies wereloaded onto Fc2, Fc3 and Fc4 of the Protein A chip (GE Healthcare,Little Chalfont, UK). IgGs were captured at a flow rate of 10 μl/min togive an immobilisation level (RL) of ˜63 RU, the theoretical value toobtain an Rmax of ˜50 RU. The surface was then allowed to stabilize.Kinetic data was obtained with factor Bb as analyte and using a flowrate of 30 μl/min to minimize any potential mass transfer effects.Multiple repeats of a blank and a repeat of a single concentration ofthe analyte were programmed into the kinetic run in order to check thestability of both the surface and analyte over the kinetic cycles. Forkinetic analysis, a two-fold dilution range was selected from 12. 5 nMto 0. 391 nM factor Bb. The association phase of factor Bb was monitoredfor 360 seconds and the dissociation phase was monitored for 600seconds. Regeneration of the Protein A surface was conducted using twoinjections of 10 mM glycine-HCL pH 1. 5. The signal from the referencechannel Fc1 was subtracted from that of Fc2, Fc3 and Fc4 to correct fordifferences in non-specific binding to a reference surface, and a globalRmax parameter was used in the 1-to-1 binding model.

The sensorgrams and fitted data for the binding of chimeric antibodiesand humanized variants to factor Bb are shown in FIGS. 4A-4C. Therelative K_(D) was calculated by dividing the K_(D) of the humanizedvariants by that of the chimeric antibody on the same chip. The kineticparameters measured for the interaction of factor Bb with chimericantibodies and humanized variants are shown in Table 10. Two humanizedvariants, VH4/Vκ6 and VH4/Vκ7 (bold), showed relative K_(D)s withintwo-fold of the reference chimeric antibody.

TABLE 10 Multiple Cycle Kinetic Parameters of the Humanized Variants andReference Antibodies Binding to Factor Bb Chi² Relative Antibody ka(1/Ms) kd (1/s) KD (M) (RU²) KD VH0/Vκ0 4.87 × 10⁵ 9.69 × 10⁻⁴ 1.99 ×10⁻⁹ 0.0515 1.00 VH4/Vκ6 3.76 × 10⁵ 8.05 × 10⁻⁴ 2.14 × 10⁻⁹ 0.0389 1.08VH4/Vκ7 3.22 × 10⁵ 8.41 × 10⁻⁴ 2.61 × 10⁻⁹ 0.645  1.31 VH0/Vκ0 5.00 ×10⁵ 9.54 × 10⁻⁴ 1.91 × 10⁻⁹ 0.033  1.00 VH6/Vκ6 2.51 × 10⁵ 1.30 × 10⁻³5.17 × 10⁻⁹ 0.0307 2.71 VH6/Vκ7 2.18 × 10⁵ 1.24 × 10⁻³ 5.70 × 10⁻⁹0.0692 2.98 VH0/Vκ0 4.95 × 10⁵ 9.40 × 10⁻⁴ 1.90 × 10⁻⁹ 0.0442 1.00VH7/Vκ7 2.22 × 10⁵ 2.11 × 10⁻³ 9.51 × 10⁻⁹ 0.0219 5.01

Factor Bb Competition ELISA

Lead purified variants and chimeric antibodies were tested for theirbinding to factor Bb using competition against murine parental antibody.

Factor Bb was diluted in 1× PBS to 1.0 μg/ml and 100 μl/well was coatedovernight at 4° C. on a 96-well ELISA plate. The following day theplates were blocked for two hours at room temperature with 1% casein/PBSbefore washing 2× with PBS pH 7.4. In a 96-well dilution plate a fixedconcentration of murine parental antibody (0.5 μg/ml, finalconcentration) was added in equal volume to a four-fold titration seriesof test antibody (starting from 45 μg/ml to 0.01 μg/ml, finalconcentration) diluted in blocking buffer. After washing the plate 3×with PBS-T, 100 μl of chimeric/test antibody mix was added to the ELISAplate. After incubating at room temperature for one hour, the plate waswashed 3× with PBS-T and 100 μl of anti-mouse IgG Fc-Specific HRP(Sigma, Dorset, UK) diluted 1:1000 in PBS-T was applied for one hour atroom temperature to detect bound mouse antibody. For color development,the plate was washed 3× with PBS-T following which 100 μl of TMBsubstrate was added and incubated for approximately five minutes at roomtemperature. The reaction was stopped with 50 μl of 3.0 M hydrochloricacid and absorbance was read immediately using a DYNEX ® plate reader at450 nm.

The results were plotted and are shown in FIG. 5. IC50 values werecalculated for each variant and relative IC50 values were calculated bydividing the IC50 of the humanized variant by that of the chimericantibody assayed on the same plate (Table 11). All lead variantsdemonstrated

IC50 values within two-fold of the parent antibody.

TABLE 11 Half Maximal Inhibitory Concentration (IC50) Values Obtainedfrom Competition ELISA of Humanized Leads to Factor Bb. IC50 RelativeIC50 Variant (nM) (nM) VH0/Vκ0 1.23 1.00 VH4/Vκ6 1.02 0.83 VH4/Vκ7 0.750.61 VH6/Vκ6 0.93 0.75 VH6/Vκ7 0.82 0.66 VH7/Vκ7 1.43 1.16

Example 2 Activity of Humanized Variants Inhibition of WIESLAB® AP byAnti-Factor Bb Humanized Variants in Human Serum

The ability of the humanized variants to inhibit complement AP activitywas measured using the Complement System Alternative Pathway WIESLAB®kit. In this plate-based assay, lipopolysaccharide (LPS) coated wellslead to specifically activation of the alternative pathway withdetection of membrane attack complex (MAC) deposition serving as thereadout. 5. 56% normal human serum (NHS) was incubated with a dilutionseries of the parental antibody and humanized variants along with ahuman IgG4 control antibody starting at 100 μg/mL. OD 405 nm wasmeasured and compared to the kit positive and negative controls. Datawere plotted relative to the plate positive control (FIG. 6A). Allhumanized variants showed inhibition of AP-mediated MAC depositionsimilar to the parental antibody in human serum (Table 13).

TABLE 12 Activity of Humanized anti-factor Bb Antibodies in WIESLAB ® APAssay. Variant AP Assay IC50 (M) VH0/Vκ0 2.4 × 10⁻⁸ VH4/Vκ6 2.2 × 10⁻⁸VH4/Vκ7 2.3 × 10⁻⁸ VH6/Vκ6 1.9 × 10⁻⁸ VH6/Vκ7 2.1 × 10⁻⁸ VH7/Vκ7 2.5 ×10⁻⁸

Inhibition of AP-Mediated Hemolysis by Humanized Variants in Human Serum

Inhibition of AP pathway-mediated hemolysis was determined using humanor cynomolgus monkey serum and rabbit erythrocytes in EGTA-containingbuffer to inhibit the classical pathway. A dilution series of theparental antibody and humanized variants starting at 200 μg/mL wereincubated with 20% human serum and 10×10⁶ rabbit red blood cells (RBCs)for one hour at 37° C. The amount of lysis was determined by measuringthe absorbance of the supernatant at 540 nm and subtracting thebackground absorbance in control wells containing ethylene diaminetetraacetic acid (EDTA). Results are shown in FIG. 6B. In FIG. 6C, theA540, representing the amount of hemolysis, is shown for each variant at100 μg/mL. VH4/VK6 and VH6/VK7 show the greatest inhibition of hemolysisas reflected by the largest reduction in A540.

Binding of The Parent Antibody and Humanized Variants to CynomolgusMonkey Factor Bb

To ensure that the humanization process did not affect speciescross-reactivity, the parental antibody and humanized derivatives weretested for binding to human and cynomolgus monkey factor Bb by BiolayerInterferometry (BLI) using an Octet Red. Briefly, biotinylatedantibodies were loaded onto SA Biosensors equilibrated in PBS, 0.1% BSA,0.02% Tween-20 (assay buffer). After a 60 second baseline in assaybuffer, antibodies were loaded onto the probes for 180 seconds, followedby another 60 second baseline. Association to human (Comptech) orcynomolgus monkey factor Bb (purified in-house) was measured for 300seconds, followed by a 300 second dissociation. Kinetic parameters werecalculated by the Octet Analysis software using a 1:1 binding model.Data are summarized in Table 13 and show that the humanization did notaffect cross-reactivity to factor Bb from cynomolgus monkey.

TABLE 13 Binding of Humanized Variants to Human and Cynomolgus Bb byBLI. KD (M) for KD (M) for Antibody Human Bb Cyno Bb Anti-factor Bbantibody in Table 1 1.9 × 10⁻⁹ 1.1 × 10⁻⁸ VH0/Vκ0 2.0 × 10⁻⁹ 1.0 × 10⁻⁸VH4/Vκ6 1.6 × 10⁻⁹ 8.6 × 10⁻⁹ VH4/Vκ7 1.9 × 10⁻⁹ 1.1 × 10⁻⁸ VH6/Vκ6 1.9× 10⁻⁹ 1.1 × 10⁻⁸ VH6/Vκ7 1.9 × 10⁻⁹ 1.1 × 10⁻⁸ VH7/Vκ7 2.9 × 10⁻⁹ 1.6 ×10⁻⁸Binding of V_(H)4/Vκ6-IgG4v2 and V_(H)6/V_(K)7-IgG4v2 to Human andCynomolgus Monkey Factor Bb

To determine whether the modification of the Fc portion of theantibodies affected affinity and species cross-reactivity,V_(H)4/V_(K)6-IgG4v2 and V_(H)6/V_(K)7-IgG4v2 were tested alongsidetheir parental antibodies, V_(H)4/V_(K)6 and V_(H)6/V_(K)7,respectively, for their ability to bind human and cynomolgus monkeyfactor Bb by BLI. VH4/VK6-IgG4v2 and VH6/VK7-IgG4v2 contain mutations inthe Fc region that increase the affinity for an Fc receptor. Theexperiment was carried out as described in the example above and resultsare summarized in Table 14. As expected, modification of the Fc did notaffect binding.

TABLE 14 Binding Humanized Anti-Factor Bb Antibodies to Human andCynomolgus Factor Bb by BLI. KD (M) for KD (M) for Antibody Human BbCyno Bb V₄6/V_(K)6-IgG4v1 1.6 × 10⁻⁹ 1.1 × 10⁻⁸ V_(H)4/V_(K)6-IgG4v2 1.6× 10⁻⁹ 1.1 × 10⁻⁸ V_(H)6/V_(K)7-IgG4v1 1.7 × 10⁻⁹ 1.0 × 10⁻⁸V_(H)6/V_(K)7-IgG4v2 1.7 × 10⁻⁹ 1.0 × 10⁻⁸Specificity of V_(H)4/V_(K)6 and V_(H)6/V_(K)7 for Factor Bb (theActivated Form of Factor B)

The ability of V_(H)4/V_(K)6 and V_(H)6/V_(K)7 to bind to zymogen factorB and factor Bb from both human and cynomolgus monkey was determined bysurface plasmon resonance using a Biacore T200. Human factor B andfactor Bb were purchased from Comptech and cynomolgus monkey factor Band factor Bb were purified in-house. Briefly, the monoclonal antibodieswere captured on a Biacore Series S Protein A chip in HBSP+ (10 mMHEPES, 150 mM NaCl, 0.05% P20 pH 7.4) at a flow rate of 30 μL/min. Afive-point concentration series of each analyte was tested for bindingusing single cycle kinetics with a contact time of 180 seconds perconcentration followed by a 600 sec dissociation at 25° C. and a flowrate of 60 μL/sec. Both human and cynomolgus monkey factor B started ata concentration of 500 nM followed by 2-fold dilutions, while human andcynomolgus monkey factor Bb started at concentrations of 30 nM and 150nM, respectively, followed by 2-fold dilutions. Data were analyzed usingthe Biacore evaluation software using a 1:1 binding model. Sensorgramsare shown in FIGS. 7A-7D. Results are summarized in Table 15. Briefly,both humanized variants show roughly 10-fold higher affinities for humanfactor Bb compared to cynomolgus monkey factor Bb. Binding to human orcynomolgus monkey factor B was barely detectable and the small signalthat was observed was dominated by non-specific binding.

TABLE 15 Specificity of Lead Humanized Variants to Human or CynomolgusMonkey Factor Bb Rmax Chi² Antibody Antigen ka (1/Ms) kd (1/s) KD (M)(RU) (RU²) V_(H)4/V_(K)6- Human Factor Bb 4.10 × 10⁵ 9.25 × 10⁻⁴ 2.26 ×10⁻⁹ 108.2 0.636 IgG4v2 Human Factor B n/a n/a n/a n/a n/a Cyno FactorBb 3.49 × 10⁵ 6.83 × 10⁻³ 1.96 × 10⁻⁸ 118.7 2.42  Cyno Factor B n/a n/an/a n/a n/a V_(H)6/V_(K)7- Human Factor Bb 2.32 × 10⁵ 1.38 × 10⁻³ 5.92 ×10⁻⁹  91.8 0.114 IgG4v2 Human Factor B n/a n/a n/a n/a n/a Cyno FactorBb 1.55 × 10⁵ 8.77 × 10⁻³ 5.65 × 10⁻⁸ 104.6 0.457 Cyno Factor B n/a n/an/a n/a n/a

Binding of Chimeric Parent and Representative Humanized Variants toVarious Complement Proteins

To ensure that humanization did not introduce non-specific binding orcross-reactivity to other complement or plasma proteins, the chimericparent antibody and a representative humanized variant, VH6/VK7-IgG4v2,were tested for binding to aCls (Comptech A104), C1r (Comptech A102), C2(Comptech A112), C2a (prepared from C2), thrombin (EMD Millipore605195), elastase (EMD Millipore 324682), factor D (Competech A136), andfactor Bb (Comptech A155). Briefly, complement and plasma proteins werecoated onto ELISA plates at 2. 5 μg/mL in PBS overnight at 4° C. Plateswere then blocked with Casein for 1 hour at room temperature and washedfour times with 1× DPBS/0.05% Tween-20 followed by a single wash with 1×DPBS. Serial dilutions starting at 50 μg/ml of biotinylated chimericparent or VH6/VK7-IgG4v2 were added to the plate inPBS/0.1%casein/0.1%Tween-20 and incubated for 2 hours at roomtemperature. Plates were washed as described and a 1:10,000 dilution ofstreptavidin-HRP (Southern Biotech 7100-05) in PBS/0.1% casein/0.1%Tween-20 was added to the plate and incubated for 30 minutes at roomtemperature. Plates were again washed and Ultra TMP ELISA (Thermo 34028)was added to the plate for 1 minute, followed by stop solution). OD 450nm was read in a plate reader and background at 620 nm was subtracted.Both the chimeric parent (FIG. 8A) and the humanized variant (FIG. 8B)show complete specificity for factor Bb.

Example 3 V_(H)6/V_(k)7-IgG4v2 Produced from CHO versus HEK Behave theSame

To determine whether V_(H)6/V_(K)7-IgG4v2 produced in HEK cells behavedthe same when produced in CHO cells, V_(H)6/V_(K)7-IgG4v2was generatedby transient expression in HEK cells or by stable expression in CHOcells using standard methods.

The complement alternative pathway (CAP) and classical pathway (CCP)activities of the antibodies were determined using the Complement SystemAlternative Pathway and Classical Pathway WIESLAB® Kits, respectively,in accordance with the manufacturer's instructions. To determine CAPactivity, antibodies were tested in 5.56% normal human serum (FIG. 9A)and 5.56% normal cynomolgus monkey serum (FIG. 9B). To determine CCPactivity, antibodies were tested in 1% normal human serum (FIG. 10A) and1% normal cynomolgus monkey serum (FIG. 10B). OD_(405nm) was measuredand results were normalized to serum activity prior to antibodyinjection. V_(H)6/V_(K)7-IgG4v2 produced in CHO and HEK showed similaractivity. IC₅₀ values for both sets of antibodies were calculated andare shown in FIGS. 10A-10B. V_(H)6/V_(K)7-IgG4v2 produced in CHO showedsimilar potency to V_(H)6/V_(K)7-IgG4v2 produced in HEK. APpathway-mediated hemolysis of V_(H)6/V_(K)7-IgG4v2produced in CHO andHEK was also compared. Hemolysis was determined using 20% normal humanserum and rabbit erythrocytes in buffer containing EGTA to inhibit theclassical pathway. The amount of lysis was determined by measuring theof the supernatant and subtracting the background absorbance in controlwells containing ethylene diamine tetraacetic acid (EDTA).V_(H)6/V_(K)7-IgG4v2 produced in CHO and HEK showed similar % hemolysis(FIG. 11).

Multicycle kinetic analysis was performed on CHO and HEK-producedV_(H)6/V_(K)7-IgG4v2 antibodies using biolayer interferometry.Antibodies were diluted to 10 μg/mL in PBS+ 0.02%. Tween20, 0.1% BSA,0.05% sodium azide and loaded onto anti-hIgG Fc sensors(pre-equilibrated in the same buffer) for 90 seconds followed by a60-minute baseline in buffer. Binding of a concentration series of humanfactor Bb (Complement Technologies, #A155), ranging from 100 nM to 0.14nM in 2-fold dilutions, was measured in a 300 second association in thesame buffer, followed by a 300 second dissociation step. The bindingcurves and fitted data, as well as calculated Kds for both antibodiesare shown in FIG. 12.

REFERENCES

-   Cao et al. (2016) Haematologica 101(11): 1319-1326-   Chothia et al. (1987) J. Mol. Biol. 196:901-917-   Bryson et el. (2010). Biodrugs 24 (1):1-8-   Dall'Acqua et al. (2006) J Biol Chem 281: 23514-24-   De Vriese et al. (2015) J Am Soc Nephrol 26: 2917-2929-   Holers (2008) Immunological Reviews 223: 300-316-   Holt et al. (2003) Trends Biotechnol. 21:484-   Gold et al. (2006) Nat. Genet. 38(4): 458-462-   Kabat et al., J. Biol. Chem. 252:6609-6616 (1977)-   Kabat et al. U. S. Dept. of Health and Human Services, “Sequences of    proteins of immunological interest” (1991)-   Lefranc et al. (2003) Developmental and Comparative Immunology 27:55

MacCallum et al. (1996) J. Mol. Biol. 262:732-745

-   Perry et al. (2008) Drugs R D 9 (6): 385-396-   Schubart et al. (2019) PNAS 116(16): 7926-7931-   Smith P et al. (2012) PNAS 109: 6181-6186-   Shields et al. (2001) J Biol Chem 276: 6591-604-   Shaner et al. (2005) Nat. Methods 2:905-909-   Thurman (2015) Am J Kidney Dis 65(1): 156-168-   U.S. Pat. No. 6,737,056-   International Publication No. WO 02/060919-   International Publication No. WO 98/23289;-   International Publication No. WO 97/34631

All publications, patents, patent applications, publication, anddatabase entries (e.g., sequence database entries) mentioned herein,e.g., in the Background, Summary, Detailed Description, Examples, and/orReferences sections, are hereby incorporated by reference in theirentirety as if each individual publication, patent, patent application,publication, and database entry was specifically and individuallyincorporated herein by reference. In case of conflict, the presentapplication, including any definitions herein, will control.

Other Embodiments

In some embodiments, the present disclosure provides: 1. A humanizedantibody that binds specifically to human complement factor Bb proteinand comprises a heavy chain variable region (VH) comprising the aminoacid sequence of SEQ ID NO: 19 and a light chain variable region (VL)comprising the amino acid sequence of SEQ ID NO: 27.

2. A humanized antibody that binds specifically to human complementfactor Bb protein and comprises a heavy chain variable region (VH)comprising the amino acid sequence of SEQ ID NO: 17 and a light chainvariable region (VL) comprising the amino acid sequence of SEQ ID NO:26.

3. The humanized antibody of paragraph 1 or paragraph 2, wherein thehumanized antibody binds specifically to the human complement factor Bbprotein with an affinity of 10-6 to10-9 M.

4. The humanized antibody of any one of paragraphs 1-3, wherein thehumanized antibody inhibits a complement pathway activity.

5. The humanized antibody of paragraph 4, wherein the complementactivity is selected from the group consisting of: AP-mediated terminalmembrane attack complex (MAC) deposition, AP-mediated hemolysis, C3fragment deposition on red blood cells or other cell types,C3b/Bb-mediated cleavage of C3, and C3bBb3b-mediated cleavage of C5.

6. The humanized antibody of any one of paragraphs 1-5, wherein thehumanized antibody is a bispecific antibody or a multispecific antibody.

7. The humanized antibody of any one of paragraphs 1-6, wherein thehumanized antibody is selected from the group consisting of an Igmonomer, a Fab fragment, a F(ab′)₂ fragment, a scFv, a scAb, and a Fv.

8. The humanized antibody of any one of paragraphs 1-6, wherein thehumanized antibody comprises a heavy chain constant region of theisotype IgG1, IgG2, IgG3, or IgG4.

9. The humanized antibody of paragraph 8, wherein the humanized antibodycomprises an IgG4 constant region or a variant thereof.

10. The humanized antibody of paragraph 9, wherein the heavy chainconstant region comprises an amino acid sequence that is at least 90%identical to any one of SEQ ID NOs: 28-30.

11. The humanized antibody of any one of paragraphs 1 and 3-10, whereinthe humanized antibody comprises a heavy chain comprising the amino acidsequence of any one of SEQ ID NOs: 32-34 and a light chain comprisingthe amino acid sequence of SEQ ID NO: 35.

12. The humanized antibody of any one of paragraphs 2-10, wherein thehumanized antibody comprises a heavy chain comprising the amino acidsequence of any one of SEQ ID

NOs: 36-38 and a light chain comprising the amino acid sequence of SEQID NO: 39.

13. A conjugate comprising the humanized antibody of any one ofparagraphs 1-12.

14. A pharmaceutical composition comprising the humanized antibody ofany one of paragraphs 1-12 or the conjugate of paragraph 13.

15. The pharmaceutical composition of paragraph 14, further comprising apharmaceutically acceptable excipient.

16. A device comprising the humanized antibody of any one of paragraphs1-12, the conjugate of paragraph 13, or the pharmaceutical compositionof paragraph 14 or paragraph 15.

17. The device of paragraph 16, wherein the device is an injectabledevice.

18. The device of paragraph 17, wherein the injectable device is asyringe, a pen, or an electronic injection device (e-Device).

19. A method of treating a subject having a complement-mediated diseaseor disorder, the method comprising: administering to the subject aneffective amount of the humanized antibody of any one of paragraphs1-13, the conjugate of paragraph 14, or the pharmaceutical compositionof paragraph 15 or 16 to treat the complement-mediated disease.

20. The method of paragraph 19, wherein the complement-mediated diseaseis selected from the group consisting of: IgA nephropathy (Berger'sdisease), atypical hemolytic uremic syndrome (aHUS), paroxysmalnocturnal hemoglobinuria (PNH), idiopathic thrombocytopenic purpura(ITP), thrombotic thrombocytopenic purpura (TTP), lupus nephritis, ANCAvasculitis, membranous nephropathy, C3 glomerulonephritis (C3GN), focalsegmental glomerulosclerosis (FSGS), multiple sclerosis, maculardegeneration, age-related macular degeneration (AMD), rheumatoidarthritis, antiphospholipid antibody syndrome, asthma,ischemia-reperfusion injury,

Type II membrano-proliferative GN, spontaneous fetal loss, Pauci-immunevasculitis, epidermolysis bullosa, recurrent fetal loss, and traumaticbrain injury.

21. A method of inhibiting a complement pathway activity in a subject,the method comprising administering to the subject an effective amountof the humanized antibody of any one of paragraphs 1-13, the conjugateof paragraph 14, or the pharmaceutical composition of paragraph 15 or 16to inhibit the complement activity.

22. The method of paragraph 21, wherein the subject has acomplement-mediated disease or disorder.

23. The method of paragraph 22, wherein the complement-mediated diseaseis selected from the group consisting of: IgA nephropathy (Berger'sdisease), atypical hemolytic uremic syndrome (aHUS), paroxysmalnocturnal hemoglobinuria (PNH), idiopathic thrombocytopenic purpura(ITP), thrombotic thrombocytopenic purpura (TTP), lupus nephritis, ANCAvasculitis, membranous nephropathy, C3 glomerulonephritis (C3GN), focalsegmental glomerulosclerosis (FSGS), multiple sclerosis, maculardegeneration, age-related macular degeneration (AMD), rheumatoidarthritis, antiphospholipid antibody syndrome, asthma,ischemia-reperfusion injury, Type II membrano-proliferative GN,spontaneous fetal loss, Pauci-immune vasculitis, epidermolysis bullosa,recurrent fetal loss, and traumatic brain injury.

24. The method of any one of paragraphs 21-23, wherein the complementactivity is selected from the group consisting of: AP-mediated terminalmembrane attack complex (MAC) deposition, AP-mediated hemolysis, C3fragment deposition on red blood cells or other cell types,C3b/Bb-mediated cleavage of C3, and C3bBb3b-mediated cleavage of C5.

25. The method of any one of paragraphs 19-24, further comprisingadministering to the subject a therapeutic agent.

26. The method of any one of paragraphs 19-25, wherein the administeringis intravenous, subcutaneous, or intramuscular.

27. A nucleic acid or nucleic acid set encoding or collectively encodingthe humanized antibody of any one of paragraphs 1-12.

28. A vector or vector set comprising the nucleic acid or nucleic acidset of paragraph 27.

29. A cell expressing the humanized antibody of any one of paragraphs1-3, the nucleic acid or nucleic acid set of paragraph 27, or the vectoror vector set of paragraph 28.

30. The cell of paragraph 29, wherein the cell is a mammalian cell.

31. The cell of paragraph 30, wherein the mammalian cell is selectedfrom the group consisting of: human embryonic kidney (HEK) cells,Chinese hamster ovary (CHO) cells, NSO myeloma cells, SP2 cells, COScells, and mammary epithelial cells.

32. A method of producing the humanized antibody, comprising culturingthe cell of any one of paragraphs 29-31 to produce the humanizedantibody.

33. The method of paragraph 32, further comprising isolating thehumanized antibody.

Equivalents and Scope

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation, many equivalents of the embodimentsdescribed herein. The scope of the present disclosure is not intended tobe limited to the above description, but rather is as set forth in theappended claims.

Articles such as “a,” “an,” and “the” may mean at least one than oneunless indicated to the contrary or otherwise evident from the context.Claims or descriptions that include “or” between two or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present, unless indicated to the contrary or otherwiseevident from the context. The disclosure of a group that includes “or”between two or more group members provides embodiments in which exactlyone member of the group is present, embodiments in which more than onemembers of the group are present, and embodiments in which all of thegroup members are present. For purposes of brevity those embodimentshave not been individually spelled out herein, but it will be understoodthat each of these embodiments is provided herein and may bespecifically claimed or disclaimed.

It is to be understood that the disclosure encompasses all variations,combinations, and permutations in which at least one limitation,element, clause, or descriptive term, from at least one of the claims orfrom at least one relevant portion of the description, is introducedinto another claim. For example, a claim that is dependent on anotherclaim can be modified to include at least one of the limitations foundin any other claim that is dependent on the same base claim.Furthermore, where the claims recite a composition, it is to beunderstood that methods of making or using the composition according toany of the methods of making or using disclosed herein or according tomethods known in the art, if any, are included, unless otherwiseindicated or unless it would be evident to one of ordinary skill in theart that a contradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that every possible subgroup of the elements is alsodisclosed, and that any element or subgroup of elements can be removedfrom the group. It is also noted that the term “comprising” is intendedto be open and permits the inclusion of additional elements or steps. Itshould be understood that, in general, where an embodiment, product, ormethod is referred to as comprising particular elements, features, orsteps, embodiments, products, or methods that consist, or consistessentially of, such elements, features, or steps, are provided as well.For purposes of brevity those embodiments have not been individuallyspelled out herein, but it will be understood that each of theseembodiments is provided herein and may be specifically claimed ordisclaimed.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” are to beunderstood to be open-ended, i.e., to mean including but not limited to.Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and/or the understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value withinthe stated ranges in some embodiments, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.For purposes of brevity, the values in each range have not beenindividually spelled out herein, but it will be understood that each ofthese values is provided herein and may be specifically claimed ordisclaimed. It is also to be understood that unless otherwise indicatedor otherwise evident from the context and/or the understanding of one ofordinary skill in the art, values expressed as ranges can assume anysubrange within the given range, wherein the endpoints of the subrangeare expressed to the same degree of accuracy as the tenth of the unit ofthe lower limit of the range.

Where websites are provided, URL addresses are provided asnon-browser-executable codes, with periods of the respective web addressin parentheses. The actual web addresses do not contain the parentheses.

In addition, it is to be understood that any particular embodiment ofthe present disclosure may be explicitly excluded from any at least oneof the claims. Where ranges are given, any value within the range mayexplicitly be excluded from any at least one of the claims. Anyembodiment, element, feature, application, or aspect of the compositionsand/or methods of the disclosure, can be excluded from any at least oneclaims. For purposes of brevity, all of the embodiments in which atleast one elements, features, purposes, or aspects is excluded are notset forth explicitly herein.

The terms “about” and “substantially” preceding a numerical value mean±10% of the recited numerical value.

What is claimed is:
 1. A humanized antibody that binds specifically tohuman complement factor Bb protein, wherein the antibody comprises aheavy chain variable region (V_(H)) comprising the amino acid sequenceof SEQ ID NO: 19 and a light chain variable region (V_(L)) comprisingthe amino acid sequence of SEQ ID NO:
 27. 2. The humanized antibody ofclaim 1, wherein the humanized antibody is a multispecific antibody. Thehumanized antibody of claim 2, wherein the multispecific antibody is abispecific antibody.
 4. The humanized antibody of claim 1, wherein thehumanized antibody is selected from the group consisting of an Igmonomer, a Fab fragment, a F(ab′)₂ fragment, a scFv, a scAb, and a Fv.5. The humanized antibody of claim 1, wherein the humanized antibodycomprises a heavy chain constant region of the isotype IgG1, IgG2, IgG3,or IgG4.
 6. The humanized antibody of claim 5, wherein the humanizedantibody comprises an IgG4 constant region.
 7. The humanized antibody ofclaim 6, wherein the IgG4 constant region comprises an S241Psubstitution and an L248E substitution, by Kabat numbering.
 8. Thehumanized antibody of claim 7, wherein the IgG4 constant region furthercomprises an M459L substitution and an N466S substitution, by Kabatnumbering.
 9. The humanized antibody of claim 1, wherein the humanizedantibody comprises a heavy chain constant region comprising the aminoacid sequence of any one of SEQ ID Nos. 28-30.
 10. The humanizedantibody of claim 1, wherein the humanized antibody comprises a lightchain constant region comprising the amino acid sequence of SEQ ID NO:31.
 11. The humanized antibody of claim 9, wherein the humanizedantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 32 and a light chain comprising the amino acid sequence ofSEQ ID NO:
 35. 12. The humanized antibody of claim 9, wherein thehumanized antibody comprises a heavy chain comprising the amino acidsequence of SEQ ID NO: 33 and a light chain comprising the amino acidsequence of SEQ ID NO:
 35. 13. The humanized antibody of claim 9,wherein the humanized antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 34 and a light chain comprising theamino acid sequence of SEQ ID NO:
 35. 14. A conjugate comprising thehumanized antibody of claim
 1. 15. The conjugate of claim 14, whereinthe humanized antibody is conjugated to a therapeutic agent.
 16. Acomposition comprising the humanized antibody of claim
 1. 17. Thecomposition of claim 16, further comprising a pharmaceuticallyacceptable excipient.
 18. A device comprising the humanized antibody ofclaim
 1. 19. A nucleic acid or nucleic acid set encoding or collectivelyencoding the humanized antibody of claim
 1. 20. A vector or vector setcomprising the nucleic acid or nucleic acid set of claim
 19. 21. Anisolated cell comprising the nucleic acid or nucleic acid set of claim19.
 22. The isolated cell of claim 21, wherein the cell is a mammaliancell.
 23. The isolated cell of claim 22, wherein the mammalian cell isselected from human embryonic kidney (HEK) cells, Chinese hamster ovary(CHO) cells, NSO myeloma cells, SP2 cells, COS cells, and mammaryepithelial cells.
 24. A method of producing a humanized antibody thatbinds specifically to human complement factor Bb protein, wherein themethod comprises culturing the isolated cell of claim 21 to produce thehumanized antibody.
 25. A method of treating a subject having acomplement-mediated disease or disorder, wherein the method comprisesadministering to the subject a therapeutically effective amount of ahumanized antibody that binds specifically to human complement factor Bbprotein, wherein the antibody comprises a heavy chain variable region(V_(H)) comprising the amino acid sequence of SEQ ID NO: 19 and a lightchain variable region (V_(L)) comprising the amino acid sequence of SEQID NO:
 27. 26. The method of claim 25, wherein the complement-mediateddisease or disorder is PNH, aHUS, IgA nephropathy, lupus nephritis, ANCAvasculitis, membranous nephropathy, or C3 glomerulonephritis (C3GN). 27.A method of inhibiting a complement pathway activity in a subject,wherein the method comprises administering to the subject a humanizedantibody that binds specifically to human complement factor Bb protein,wherein the antibody comprises a heavy chain variable region (V_(H))comprising the amino acid sequence of SEQ ID NO: 19 and a light chainvariable region (V_(L)) comprising the amino acid sequence of SEQ ID NO:27.
 28. A humanized antibody that binds specifically to human complementfactor Bb protein and comprises a heavy chain variable region (V_(H))comprising the amino acid sequence of SEQ ID NO: 17 and a light chainvariable region (V_(L)) comprising the amino acid sequence of SEQ ID NO:26.
 29. A method of treating a subject having a complement-mediateddisease or disorder, wherein the method comprises administering to thesubject a therapeutically effective amount of the humanized antibody ofclaim
 28. 30. A method of inhibiting a complement pathway activity in asubject, wherein the method comprises administering to the subject thehumanized antibody of claim 28.